Detection of cancer by elevated levels of BCL-2

ABSTRACT

The present invention relates to a method for the diagnosis, prognosis, and monitoring of cancer, such as early or late stage ovarian cancer, in a subject by detecting Bcl-2 in a biological sample from the subject, preferably a urine or blood sample. Bcl-2 may be measured using an agent that detects or binds to Bcl-2 protein or an agent that detects or binds to encoding nucleic acids, such as antibodies specifically reactive with Bcl-2 protein or a portion thereof. The invention further relates to kits for carrying out the methods of the invention. The invention further relates to a device for the rapid detection of Bcl-2 in a bodily fluid and methods for rapidly measuring Bcl-2 in a bodily fluid.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/771,677, filed Feb. 9, 2006, which is herebyincorporated by reference herein in its entirety, including any figures,tables, nucleic acid sequences, amino acid sequences, and drawings.

This invention was made with government support under US Army Departmentof Defense New Idea Award #W81XWH-07-1-0276(PAK). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer markers are substances that can be found in the body (usually inthe blood or urine) when cancer is present. They can be products of thecancer cells themselves, or of the body in response to cancer or otherconditions. For several reasons, cancer markers themselves are usuallynot enough to diagnose (or rule out) a specific type of cancer. Mostcancer markers can be produced by normal cells as well as by cancercells, even if in smaller amounts. Sometimes, non-cancerous diseases canalso cause levels of certain cancer markers to be higher than normal.Further, not every person with cancer may have higher levels of a cancermarker. For these reasons, only a small amount of cancer markers arecommonly used by most doctors. When a doctor does look at the level of acertain cancer marker, he or she will typically consider it along withthe results of the patient's history and physical exam, and other labtests or imaging tests.

Screening refers to looking for cancer in individuals who have nosymptoms of the disease, while early detection is finding cancer at anearly stage of the disease, when it is less likely to have spread (andis more likely to be treated effectively). Although cancer markers wereoriginally investigated and developed to test for cancer in peoplewithout symptoms, very few markers have been shown to be helpful in thisway.

Ovarian cancer has the highest mortality among gynecological cancers.The lack of early symptoms and the absence of a reliable screening testto detect ovarian cancer result in over 70% of women being diagnosedafter the disease has spread beyond the ovary so that the prognosis ispoor with approximately 12,000 deaths due to ovarian cancer annually(5-year survival is no better than 37%). Currently, physical pelvicexamination by a physician, ultrasound or measuring blood levels forCA125 are the only standard methods available for detection of ovariancancer. However, none of these methods provides a reliably consistentand accurate method to detect ovarian cancer. For example, while over80% of women with ovarian cancer will have elevated blood levels ofCA125, blood levels of CA125 are only about 50% accurate for detectingearly stage disease. The development of an alternate and new test toreliably and accurately detect all ovarian cancers is imperative. Thus,what is needed is a technology that overcomes the current lack of areliable, accurate, safe and cost-effective test for ovarian cancer.Furthermore, what is needed is a technology that accurately detects allovarian cancers, many of which now go undetected, as well as monitordisease burden throughout the course of ovarian cancer.

An accurate, safe, simple, and reliable test to diagnosis ovarian cancerwould benefit all women, in the United States and worldwide, includingmedically underserved geographical areas and especially women at highrisk for developing ovarian cancer. Given that approximately 25,000women are diagnosed with ovarian cancer annually in the U.S., abiomarker of ovarian cancer that is detectable in both early and latestages of disease would not only confirm the diagnosis of ovariancancer, but could also potentially detect thousands of previouslyundiagnosed ovarian cancers. This is especially important for detectionof ovarian cancer in early stages where the disease is confined to theovary, but currently accounts for less than 10% of diagnosed ovariancancers. In these situations, surgical debulking of the diseased ovaryincreases patient survival to over 90% and would be expected to reducemedical costs. The ability to accurately detect and monitor ovariancancer in each patient through the course of her disease, would not onlyserve for initial ovarian cancer diagnosis, but would also indicatetherapeutic efficacy and/or recurrent disease. The development of acommercially available, FDA-approved ELISA-based test, for example,could become the gold standard for clinical diagnosis of ovarian cancer.

While apoptosis is an essential biological process for normaldevelopment and maintenance of tissue homeostasis, it is also involvedin a number of pathologic conditions including tissue injury,degenerative diseases, immunological diseases and cancer (Lowe, S. W.and Lin, A. W. Carcinogenesis, 2000, 21:485-495). Whether activated bymembrane bound death receptors (Ashkenazi, A. et al. J. Clin. Invest.,1999, 104:155-162; Walczak, H. Krammer, P. H. Exp. Cell Res, 2000,256:58-66) or by stress-induced mitochondrial perturbation withsubsequent cytochrome c release (Loeffler, M. and Kroemer, G. Exp. CellRes., 2000, 256:19-26; Wernig, F. and Xu, Q. Prog. Biophys. Mol. Biol.,2002, 78:105-137; Takano, T. et al. Antiox. Redox. Signal, 2002,4:533-541), activation of downstream caspases leads to stepwise cellulardestruction by disrupting the cytoskeleton, shutting down DNAreplication and repair, degrading chromosomal DNA, and, finally,disintegrating the cell into apoptotic bodies (Nagata, S. Exp. CellRes., 2000, 256:12-18). The key regulators of apoptosis include membersof the bcl-2 protein family (Farrow, S. N. and Brown, R. Curr. Opin.Gen. Dev., 1996, 6:45-49).

The bcl-2 protein family consists of both pro- and anti-apoptoticprotein family members that act at different levels of the apoptoticcascade to regulate apoptosis. The bcl-2 family members contain at leastone Bcl-2-homology (BH) domain (Farrow, S. N. and Brown, R. Curr. Opin.Gen. Dev., 1996, 6:45-49). Though all bcl-2 family members demonstratemembrane channel forming activity, Bcl-2 (the archetypal bcl-2 familymember) channels are cation (Ca⁺⁺) selective and, owing to its exclusiveER and mitochondrial membrane localization (Thomenius, M. J. andDistelhorst, C. W. J. Cell Sci., 2003, 116:4493-4499), theanti-apoptotic function of Bcl-2 is at least partly mediated by itsability to prevent calcium release from the ER and subsequentmitochondrial membrane perturbation and cytochrome c release. SinceBcl-2 is overexpressed in many tumor types including ovarian cancer(Sharma, H. et al. Head Neck, 2004, 26:733-740; Hanaoka, T. et al. Intl.J. Clin. Oncol., 2002, 7:152-158; Trisciuoglio, D. et al. J. CellPhysiol., 2005, 205:414-421; Khalifeh, I. et al. Int. J. Gynecol.Pathol., 2004, 23:162-169; O'Neill, C. J. et al. Am. J. Surg. Pathol.;2005, 29:1034-1041), it contributes to chemoresistance by stabilizingthe mitochondrial membrane against apoptotic insults. Currently,preclinical studies focus on the development of agents to inhibit Bcl-2,including antisense oligonucleotides such as G3,139 (Ackermann, E. J. etal. J. Biol. Chem., 1999, 274:11245-11252), and small molecularinhibitors of Bcl-2 (Lickliter, J. D. et al. Leukemia, 2003,17:2074-2080). Though such studies target Bcl-2 for therapeuticintervention, quantification of urinary Bcl-2 has not previously beenreported in the literature.

It would be advantageous to have available assays that provide safe,sensitive, specific, and economical methods for the detection of cancerssuch as ovarian cancer, which would benefit society worldwide.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to cancer screening. Bcl-2 constitutes abiomarker for prognosis, diagnosis, and monitoring of cancer, such asreproductive cancer. For example, Bcl-2 may be used to diagnose andmonitor early stage and late stage ovarian cancer. Bcl-2 may be used asa biomarker for cancer before surgery and after relapse. Bcl-2, andagents that bind Bcl-2 polynucleotides or polypeptides may be used todetect and monitor ovarian cancer, and other reproductive ornon-reproductive cancers.

Thus, more particularly, this invention relates to the detection ofcancer by screening for elevated levels of Bcl-2 in biological samples,such as urine, blood (e.g., whole blood, serum, or plasma), and ascitesfluid. In one embodiment, the cancer is ovarian cancer. In anotherembodiment, the cancer is a type selected from the group consisting ofbreast, endometrial, cervical, lung, colon, prostate, melanoma,glioblastoma, sarcoma, bladder, and head and neck. Optionally, themethod further comprises verifying that the subject is suffering fromthe cancer detected (e.g., by assessing for the presence of one or morecancer symptoms, detecting additional cancer markers, detecting thepresence of the cancer through an imaging modality such as X-ray, CT,nuclear imaging (PET and SPECT), ultrasound, MRI) and/or treating thesubject for the cancer detected (e.g., by surgery, chemotherapy, and/orradiation).

The invention also relates to kits for carrying out the methods of theinvention.

In another aspect, the present invention relates to a device for therapid detection of Bcl-2 in a bodily fluid such as blood or urine.Preferably, the device is a lateral flow device. In one embodiment, thedevice comprises an application zone for receiving a sample of bodilyfluid such as blood or urine; a labeling zone containing a binding agentthat binds to Bcl-2 in the sample; and a detection zone whereBcl-2-bound binding agent is retained to give a signal, wherein thesignal given for a sample from a subject with a Bcl-2 level lower than athreshold concentration is different from the signal given for a samplefrom a patient with a Bcl-2 level equal to or greater than a thresholdconcentration.

In another aspect, the invention relates to a simple, rapid, reliable,accurate and cost effective test for Bcl-2 in a bodily fluid such asblood or urine, similar to currently available in-home pregnancy teststhat could be used by subjects at home, in a physicians' office, or at apatient's bedside.

In one embodiment, the test is a method for measuring Bcl-2 in a bodilyfluid, comprising: (a) obtaining a sample of bodily fluid, such as bloodor urine, from a subject; (b) contacting the sample with a binding agentthat binds to any Bcl-2 in the sample; (c) separating Bcl-2-boundbinding agent; (d) detecting a signal associated with the separatedbinding agent from (c); and (e) comparing the signal detected in step(d) with a reference signal which corresponds to the signal given by asample from a subject with a Bcl-2 level equal to a thresholdconcentration. In one embodiment, the bodily fluid is urine, and thethreshold concentration is between 0 ng/ml and 2.0 ng/ml. In anotherembodiment, the bodily fluid is urine, and the threshold concentrationis 1.8 ng/ml.

To assess whether urinary levels of Bcl-2 could be used to detectovarian cancer, urine was collected from normal healthy volunteers, frompatients with ovarian cancer and measured for Bcl-2 by ELISA. Theaverage amount of Bcl-2 in the urine of cancer patients was generally atleast 10× greater than healthy controls. In addition, none of the urinesamples collected from 35 women with benign gynecologic disease(including teratomas, ovarian cysts, leiomyomas, polycystic ovariandisease, adenofibromas or cystadenomas) had Bcl-2 levels above thatfound in normal, healthy volunteers. Urinary levels of Bcl-2 decreasedup to 100% in ovarian cancer patients following debulking surgery. Thesensitivity and specificity for elevated urinary Bcl-2 associated withovarian cancer was almost 100% while blood levels of CA125>35 U/ml onlyidentified 68% of ovarian cancer patients. Comparison of clinicalparameters indicated that urinary levels of Bcl-2 correlated well withtumor stage and grade. However, urinary levels of Bcl-2 were not relatedto patient age or tumor size. Therefore, quantification of urinary Bcl-2by ELISA-based assays provides a safe, sensitive, specific andeconomical method to detect ovarian cancer, to monitor ovarian cancerthroughout the course of disease and to predict therapeutic andprognostic outcome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram depicting urinary levels of Bcl-2. Urinary levelsof Bcl-2 are higher in patients with ovarian cancer compared with normalhealthy volunteers. Urine was collected from normal healthy volunteersand from patients with ovarian cancer (including histological subtypesserous, mucinous) and peritoneal cancer. Serous ovarian cancers werefurther subdivided into stage 1 (the first three bars on the left in theserous grouping), stage 2 (the next eight bars in the serous grouping(i.e., bars 4-11 from the left of the serous grouping)) and stage 3 (theeleven bars in the right-hand section of the serous grouping (i.e. bars12-22 from the left of the serous grouping). The urine was tested intriplicate for Bcl-2 by ELISA (ELISA kits from Bender MedSystems,catalog #BMS244/3) and the results expressed as the average ng/mlBcl-2±S.E. The data indicate consistently elevated levels of Bcl-2 inthe urine of patients with cancer. Student t-test analysis revealed astatistical difference between normal and cancer specimens at p<0.00001.

FIG. 2 is a histogram depicting urinary levels of Bcl-2 in normal andcancer patients. Additional urine specimens were collected from normalhealthy volunteers and from patients with ovarian cancer (includinghistological subtypes endometroid, serous and mucinous) and peritonealcancer. Serous ovarian cancers were further subdivided into stage 1 (7left-most bars in serous grouping), stage 2 (bars 8-17 from the left inthe serous grouping) and stage 3 (12 right-most bars in serousgrouping). The urine was tested in triplicate for Bcl-2 by ELISA (ELISAkits from Bender Med Systems), the results expressed as the averageng/ml Bcl-2 and represent all the normal and the pre-surgical cancerurine specimens tested to date. In agreement with FIG. 1, the dataindicate consistently elevated levels of Bcl-2 in the urine of patientswith cancer. Student t-test analysis reveal a statistical differencebetween normal and cancer specimens at p<0.00001.

FIGS. 3A and 3B are histograms demonstrating that urinary Bcl-2 isrelated to tumor stage and grade, respectively. Levels of urinary Bcl-2were plotted against tumor stage from all available histological ovariancancer subtypes (serous, endometriod, mucinous). Stages I, II, III and Vare represented by Roman numerals with groupings underneath. Thoughstill considerably higher than normal controls, FIG. 3A illustrates thaturinary levels of Bcl-2 were lowest in stage I and II tumors (averageng/ml Bcl-2=2.2) where the disease is localized within the ovary andperitoneal cavity, respectively. Urinary levels of Bcl-2 were greatestamong stage III and V (average ng/ml Bcl-2=4.22) when the disease hasspread well beyond the ovary or is recurrent disease, respectively.

FIGS. 4A and 4B are a pair of histograms depicting the ability ofurinary Bcl-2 (FIG. 4A) relative to the measurements of plasma levels ofCA125 (FIG. 4B) in detecting ovarian cancer. Wherever possible, levels,of urinary Bcl-2 as previously shown in FIGS. 1-3 were compared withplasma levels of CA125 from the same normal healthy volunteers andcancer patients. The latter group included patients with mucinousovarian cancer (Muc), primary peritoneal cancer (PP) and serous ovariancancer (Serous). CA125 levels were determined by ELISA (kits fromBio-Quant, San Diego, Calif., Catalog #BQ1013T) in triplicate. The dataare expressed as the average ng/ml Bcl-2 (A) and average U/ml CA125(FIG. 4B). The sensitivity and specificity to detect ovarian cancer byelevated levels of urinary Bcl-2 was almost 100%. In contrast, CA125blood levels>35 U/ml, the current standard for ovarian cancer detection,only correctly identified 68% of ovarian cancer patients.

FIG. 5 is a histogram showing that urinary Bcl-2 does not correlate withpatient age. To examine whether elevated urinary levels of Bcl-2 incancer patients correlated with patient age, levels of urinary Bcl-2 (asdetermined previously in FIGS. 1-3 and 4A-4B) were compared againstpatient age in years. Though the average age of normal healthyvolunteers in this study was somewhat lower (54.8 years) than cancerpatients (66.2 years), there was no statistical difference in agebetween the groups due to the wide range in age (see insert in FIG. 5).In addition, the average age of cancer patients is in agreement with theliterature and clinical data indicating that ovarian cancer generallytargets peri- and post-menopausal women. However, there did not appearto be a correlation between urinary levels of Bcl-2 with patient age.

FIG. 6 is a histogram showing that urinary Bcl-2 does not correlate withovarian tumor size. To examine whether elevated urinary levels of Bcl-2in cancer patients correlated with tumor size, levels of urinary Bcl-2(as determined previously in FIGS. 1-3 and 4A-4B) were compared againsttumor size. Tumors were grouped as: 1=microscopic in size; 3=tumors lessthan 3 cm; 6=tumors between 3 and 6 cm; 10=tumors greater than 6 cm andup to 10 cm; 11=tumors greater than 10 cm. The data indicate that theredid not appear to be a correlation between urinary levels of Bcl-2 withtumor size.

FIGS. 7A and 7B are a pair of histograms showing that urinary Bcl-2decreases after ovarian cancer debulking surgery. To further test theaccuracy of urinary Bcl-2 to detect ovarian cancer, levels of urinaryBcl-2 were compared in those available ovarian cancer patientsimmediately prior to (black bars) and within 2 weeks following (greybars) initial debulking surgery (removal of all visible tumor) (FIG.7A). For those 7 patients where urine samples were collected before andafter initial surgery, Bcl-2 levels decreased up to 100% followingsurgical removal of tumor. These data, then, suggest that the tumor isthe source of Bcl-2 found elevated in the urine of patients with ovariancancer and that levels of urinary Bcl-2 parallel the presence of ovariancancer. In addition, urine samples were collected from 5 of the 7patients in FIG. 7A on subsequent follow up clinical visits ranging from7 to 11 months following initial surgery and measured for Bcl-2 (bluebars) (FIG. 7B). Urinary Bcl-2 levels remained low in 3 follow-uppatients (#41, 43, 54) and became elevated in 2 patients (#5, 27).Preliminary chart review indicated that patients #41, 43, 54 wereundergoing chemotherapy at the time of follow-up visits and that theirovarian cancer disease was under control. In contrast, chart reviewsuggests that patients #5, 27 had recurrent disease (5B, 27B) and thatpatient #27b underwent additional tumor debulking surgery. In agreementwith the clinical information, urinary Bcl-2 levels remained reduced inpatients undergoing chemotherapy and who had no apparent or minimalresidual disease (#41, 43, 54). Likewise, elevated urinary Bcl-2 levelscorrelated with the presence of recurrent disease (#5b, 27B) anddecreased with subsequent disease debulking (#27c).

FIGS. 8A and 8B show results of Bcl-2 testing in patients with benigngynecologic disease. Urinary samples were examined by ELISA for Bcl-2 inpatients with benign gynecologic disease. Samples were examined intriplicate and the data expressed as the average ng/ml of Bcl-2±S.E(FIG. 8A). Benign gynecologic disease samples were subdivided by type(benign cystic teratoma, simple cyst, leiomyoma, polycystic ovary,adenofibroma, mucinous and serous cystadenoma) with ovarian cancerpatient sample #41 (white bar) serving as an internal positive control.Average urinary Bcl-2 ng/ml±S.E. among benign disease are indicted belowtheir respective heading. Samples from FIG. 2 and FIG. 3A werere-plotted to show distribution of Bcl-2 expression for this study group(n=92), shown in FIG. 8B. Bcl-2 levels in benign, cancer and normalindividuals ranged from 0.115-1.016 ng/ml, 1.12-9.8 ng/ml and 0-1.26ng/ml and averaged 0.614 ng/ml, 3.4 ng/ml and 0.21 ng/ml, respectively.

FIG. 9 is a histogram showing that Bcl-2 can be secreted into cellculture conditioned medium. Conditioned medium (CM) was collected fromestablished cancer cell lines representing ovarian (OV2008, SKOV3, PA1),cervical (Hela), prostate (LNCap, DU145, PC-3), head and neck (HN5a) andlymphoma (Raji) cancers and examined by ELISA for presence of Bcl-2.Data are expressed as the mean of triplicate samples. The presence ofBcl-2 in the CM of ovarian, cervical and prostate cancer cell culturessuggests that these cancer cells produce and secrete Bcl-2.

FIG. 10 shows that Bcl-2 is over-expressed in some cancers cells. Celllysates from established cancer cell lines representing ovarian (SW626,C13), head and neck (HN5a), cervical (Hela) and prostate (DU145) cancerswere western immunoblotted for Bcl-2. Actin served as a loading controland FHIOSE118 cells (SV-40 large T antigen transfected human ovariansurface epithelial cells) served as a normal, non-malignant ovariansurface epithelial control cells. Following densitometric analyses, thelevel of Bcl-2 was normalized to actin, noted below the blots. Normalcells contained negligible amounts of bcl-2 while ovarian and cervicalcancer cells contained the greatest amount of Bcl-2.

FIG. 11 shows Bcl-2 protein concentrations following storage. Urinesamples from normal healthy individuals (#506, 508) and patients withovarian cancer (#77, 97) were originally tested for Bcl-2 as part ofthis study (control) and following storage for 4 days at either roomtemperature (25° C.), in a fridge (4° C.), in a −20° C. freezer (−20°C.) or in a −80° C. freezer (−80° C.). All samples were tested induplicate for urinary levels of Bcl-2 using an ELISA kit (BenderMedSystems).

FIG. 12 shows Bcl-2 protein concentrations in conditioned medium (CM) ofcancer cell lines following treatment with lysophosphatidic acid (LPA),including DU145, a prostate cancer cell line. The figure shows thattreatment with LPA, which often feeds back into cancer cells in themanner of an autocrine loop, stimulates secretion of Bcl-2 into the CMof some cancer cell types. As these cancer cell lines secrete Bcl-2 intotheir CM, as do ovarian cancer cell lines, the in vivo tumorcounterparts of such cell lines potentially secrete Bcl-2 intobiological fluids such as urine and/or blood and may be detected usingthe present invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is human Bcl-2 DNA (GenBank accession no. M14745); codingregion (CDS): bases 32-751.

SEQ ID NO:2 is human Bcl-2 protein (GenBank accession no. AAA35591).

SEQ ID NO:3 is human Bcl-2 DNA, transcript variant alpha (GenBankaccession no. NM_(—)000633); CDS: bases 494-1213.

SEQ ID NO:4 is human Bcl-2 protein, transcript variant alpha (GenBankaccession no. NP_(—)000624).

SEQ ID NO:5 is human Bcl-2 DNA, transcript variant beta (GenBankaccession no. NM_(—)000657); CDS: bases 494-1111.

SEQ ID NO:6 is human Bcl-2 protein, transcript variant beta (GenBankaccession no. NP_(—)000648).

DETAILED DESCRIPTION OF THE INVENTION

Bcl-2 is an effective molecular marker for cancer such as ovariancancer. Cancer markers (also called tumor markers) are molecules such ashormones, enzymes, and immunoglobulins found in the body that areassociated with cancer and whose measurement or identification is usefulin patient diagnosis or clinical management. They can be products of thecancer cells themselves, or of the body in response to cancer or otherconditions. Most cancer markers are proteins. Some cancer markers areseen only in a single type of cancer, while others can be detected inseveral types of cancer. As with other cancer markers, Bcl-2 can be usedfor a variety of purposes, such as: screening a healthy population or ahigh risk population for the presence of cancer; making a diagnosis ofcancer or of a specific type of cancer, such as ovarian cancer;determining the prognosis of a subject; and monitoring the course in asubject in remission or while receiving surgery, radiation,chemotherapy, or other cancer treatment.

To assess whether urinary levels of Bcl-2 could be used to detectovarian cancer, urine was collected from normal healthy volunteers(N=21) and from patients with ovarian (N=34) and primary peritoneal(N=2) cancer and measured in triplicate for Bcl-2 using commerciallyavailable ELISA kits (BenderMedSystems, catalog #BMS244/3) according tothe manufacturer's instructions. The results were expressed as theaverage ng/ml Bcl-2±S.E. The average amount of Bcl-2 in the urine ofhealthy volunteers was 0.204 ng/ml while that from pre-surgical patientswith cancer averaged 3.12 ng/ml, generally at least 10× greater thanthat found in normal controls. Student t-test analysis revealed astatistical difference between normal and cancer specimens at p<0.00001.Comparison of clinical parameters indicated that urinary levels of Bcl-2correlated well with tumor stage and grade (FIGS. 3A and 3B).

Plasma samples from some of these same individuals above were examinedin triplicate for CA125 levels by a commercially available ELISA(Bio-Quant, catalog #BQ1013T) according to the manufacturer'sinstructions. The sensitivity and specificity for elevated urinary Bcl-2associated with ovarian cancer detection was almost 100% while bloodlevels of CA125>35 U/ml, the current standard for ovarian cancerdetection, only correctly identified 68% of ovarian cancer patients.

To further test the accuracy for levels of urinary Bcl-2 to detectovarian cancer, levels of urinary Bcl-2 were compared in those availableovarian cancer patients immediately prior to and within 2 weeksfollowing initial debulking surgery (removal of all visible tumor). Forthose 7 patients where urine samples were collected before and afterinitial surgery, Bcl-2 levels decreased up to 100% following surgicalremoval of tumor. These data, then, suggest that the tumor is the sourceof Bcl-2 found elevated in the urine of patients with ovarian cancer. Inaddition, urine samples were collected from 5 of these 7 patients onsubsequent follow up clinical visits ranging from 7 to 11 monthsfollowing initial surgery and measured for Bcl-2. Urinary Bcl-2 levelsremained low in 3 follow-up patients (#41, 43, 54) and became elevatedin 2 patients (#5, 27). Preliminary chart review indicated that patients#41, 43, and 54 were undergoing chemotherapy at the time of follow-upvisits and that their ovarian cancer disease was under control. Incontrast, chart review suggests that patients #5, 27 had recurrentdisease and that patient #27 underwent additional tumor debulkingsurgery. In agreement with the clinical information, urinary Bcl-2levels remained reduced in patients undergoing chemotherapy and who hadno apparent or minimal residual disease (#41,43,54). Likewise, elevatedurinary Bcl-2 levels correlated with the presence of recurrent disease(#5, 27) and decreased with subsequent disease debulking (#27c).

Taken together, these data indicate that quantification of urinary Bcl-2by ELISA-based assays appears to provide a novel, safe, sensitive,specific and economical method for the detection of ovarian cancer.Further, urinary levels of Bcl-2 can be used to monitor the presence ofovarian cancer throughout the course of disease and may predicttherapeutic and prognostic outcome.

In one aspect, the invention includes a method for detecting cancer in asubject, comprising detecting the presence of Bcl-2 in a biologicalsample from the subject, such as urine, blood, peritoneal fluid, orascites fluid, and wherein a level of Bcl-2 above a pre-determinedthreshold is indicative of cancer in the subject. Preferably, thedetecting is not carried out by a qualitative slot-blot assay (such asthat commercially available from BioRad).

The cancer detecting and/or monitoring using the methods, devices, andkits of the invention include, but are not limited to, breast cancer(e.g., infiltrating (invasive), pre-invasive, inflammatory, Paget'sDisease, metastatic, or recurrent); gastrointestinal/digestive cancer(e.g., appendix, bile duct, colon, esophageal, gallbladder, gastric,intestinal, liver, pancreatic, rectal, and stomach);genitourinary/urinal cancer (e.g., adrenal, bladder, kidney, penile,prostate, testicular, and urinary); gynecological cancer (e.g.,cervical, endometrial, fallopian tube, ovarian, uterine, vaginal, andvulvar); head and neck cancer (e.g., eye, head and neck, jaw, laryngeal,nasal cavity, oral cancer, pharyngeal, salivary gland, sinus, throat,thyroid, tongue, and tonsil); hematological/blood cancer (e.g.,Hodgkin's disease, leukemia (acute lymphocytic leukemia, acutegranulocytic leukemia, acute myelogenous leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia), multiple myeloma, lymphoma, andlymph node); musculoskeletal/soft tissue cancer (e.g., bone,osteosarcoma, melanoma, skin (basal cell, squamous cell), sarcoma(Ewing's sarcoma, Kaposis sarcoma)); neurological cancer (e.g., brain(astrocytoma, glioblastoma, glioma), pituitary gland, spinal cord)); andrespiratory/lung cancer (e.g., lung, (adenocarcinoma, oat cell,non-small cell, small cell, squamous cell) and mesothelioma). In oneembodiment, the cancer is ovarian cancer. In another embodiment, thecancer is a type selected from the group consisting of breast cancer,endometrial cancer, cervical cancer, lung cancer, colon cancer, prostatecancer, melanoma, glioblastoma, sarcoma, bladder cancer, and head andneck cancer.

In one embodiment of the method of the invention, the detectingcomprises: (a) contacting the biological sample with a binding agentthat binds Bcl-2 protein to form a complex; and (b) detecting thecomplex; and correlating the detected complex to the amount of Bcl-2protein in the sample, wherein the presence of elevated Bcl-2 protein isindicative of cancer. In a specific embodiment, the detecting of (b)further comprises linking or incorporating a label onto the agent, orusing ELISA-based immunoenzymatic detection.

Optionally, the methods of the invention further comprise detecting abiomarker of cancer in the same biological sample or a differentbiological sample obtained from the subject, before, during, or aftersaid detecting of Bcl-2. In one embodiment, the biomarker of cancer is abiomarker of reproductive cancer, such as gynecological cancer. Inanother embodiment, the biomarker is CA125 or OVXI. The subject may haveelevated CA125 level in the blood at the time the detecting of Bcl-2 iscarried out, or the subject may not have an elevated CA125 level in theblood at the time the detecting of Bcl-2 is carried out.

In some embodiments, the subject is suffering from cancer, such asovarian cancer, and the detecting is performed at several time points atintervals, as part of a monitoring of the subject before, during, orafter the treatment of the cancer.

Optionally, the methods of the invention further comprise comparing thelevel of Bcl-2 in the biological sample with the level of Bcl-2 presentin a normal control sample, wherein a higher level of Bcl-2 in thebiological sample as compared to the level in the normal control sampleis indicative of cancer such as ovarian cancer.

In some embodiments, the subject exhibits no symptoms of cancer at thetime the detecting of Bcl-2 is carried out. In other embodiments, thesubject exhibits one or more symptoms of cancer at the time thedetecting of Bcl-2 is carried out. For example, with respect togynecological cancer (e.g., ovarian cancer), the one or more symptoms ofgynecological cancer include those selected from the group consisting ofpelvic pain, abnormal vaginal bleeding, abdominal swelling or bloating,persistent back pain, persistent stomach upset, change in bowel orbladder pattern (such as constipation, diarrhea, blood in the stools,gas, thinner stools, frequency or urgency of urination, constipation),pain during intercourse, unintentional weight loss of ten or morepounds, vulva or vaginal abnormality (such as blister, change in skincolor, or discharge), change in the breast (such as a lump, soreness,nipple discharge, dimpling, redness, or swelling), and fatigue.

In another embodiment, the invention includes a method for prognosticevaluation of a subject having, or suspected of having, cancer,comprising: a) determining the level of Bcl-2 in a biological sampleobtained from the subject, such as urine, blood, or ascites fluid; b)comparing the level determined in step (a) to a range of Bcl-2 known tobe present in a biological sample obtained from a normal subject thatdoes not have cancer; and c) determining the prognosis of the subjectbased on the comparison of step (b), wherein a high level of Bcl-2 instep (a) indicates an aggressive form of cancer and, therefore, a poorprognosis.

The terms “detecting” or “detect” include assaying or otherwiseestablishing the presence or absence of the target Bcl-2 (Bcl-2 encodingnucleic acid sequence or Bcl-2 gene product (polypeptide)), subunitsthereof, or combinations of agent bound targets, and the like, orassaying for, interrogating, ascertaining, establishing, or otherwisedetermining one or more factual characteristics of gynecological cancer,metastasis, stage, or similar conditions. The term encompassesdiagnostic, prognostic, and monitoring applications for Bcl-2 and othercancer biomarkers. The term encompasses quantitative, semi-quantitative,and qualitative detection methodologies. In embodiments of the inventioninvolving detection of Bcl-2 protein (as opposed to nucleic acidmolecules encoding Bcl-2 protein), the detection method is preferably anELISA-based method. Preferably, in the various embodiments of theinvention, the detection method provides an output (i.e., readout orsignal) with information concerning the presence, absence, or amount ofBcl-2 in a sample from a subject. For example, the output may bequalitative (e.g., “positive” or “negative”), or quantitative (e.g., aconcentration such as nanograms per milliliter).

In an embodiment, the invention relates to a method for detecting cancerin a subject by quantitating Bcl-2 protein or encoding nucleic acids(DNA or RNA) in a biological sample such as urine from the subject,comprising (a) contacting (reacting) the biological sample with anantibody specific for Bcl-2 which is directly or indirectly labeled witha detectable substance; and (b) detecting the detectable substance.

In an embodiment, the invention relates to a method for diagnosingand/or monitoring cancer in a subject by quantitating Bcl-2 in abiological sample, such as urine or blood, from the subject, comprising(a) reacting the biological sample with an antibody specific for Bcl-2which is directly or indirectly labeled with a delectable substance; and(b) detecting the detectable substance.

Embodiments of the methods of the invention involve (a) contacting abiological sample from a subject with an antibody specific for Bcl-2which is directly or indirectly labeled with an enzyme; (b) adding asubstrate for the enzyme wherein the substrate is selected so that thesubstrate, or a reaction product of the enzyme and substrate, formsfluorescent complexes; (c) quantitating Bcl-2 in the sample by measuringfluorescence of the fluorescent complexes; and (d) comparing thequantitated levels to that of a standard.

A preferred embodiment of the invention comprises the following steps:

(a) incubating a biological sample with a first antibody specific forBcl-2 which is directly or indirectly labeled with a detectablesubstance, and a second antibody specific for Bcl-2 which isimmobilized;

(b) separating the first antibody from the second antibody to provide afirst antibody phase and a second antibody phase;

(c) detecting the detectable substance in the first or second antibodyphase thereby quantitating Bcl-2 in the biological sample; and

(d) comparing the quantitated Bcl-2 with a standard.

A standard used in a method of the invention may correspond to Bcl-2levels obtained for samples from healthy control subjects, from subjectswith benign disease (e.g., benign gynecological disease), subjects withearly stage gynecological cancer, or from other samples of the subject.Increased levels of Bcl-2 as compared to the standard may be indicativeof cancer, such as early or late stage ovarian cancer.

The invention also contemplates using the methods, devices, and kitsdescribed herein in conjunction with one or more additional markers(“biomarkers”) for cancer. Therefore, the invention contemplates amethod for analyzing a biological sample for the presence of Bcl-2 andanalyzing the same sample, or another biological sample from the samesubject, for other markers that are specific indicators of a cancer. Theone or more additional markers may be detected before, during, and/orafter detection of Bcl-2 is carried out. Examples of markers includeCA125, LPA, and OVXI. In a preferred embodiment, the markers are Bcl-2and CA125. The methods, devices, and kits described herein may bemodified by including agents to detect the additional markers, ornucleic acids encoding the markers.

Cancer markers that may be used in conjunction with the inventioninclude, but are not limited to: alpha fetoprotein (AFP), e.g., forpancreatic, kidney, ovarian, cervical, and testicular cancers;carcinogenic embryonic antigen (CEA), e.g., for lung, pancreatic,kidney, breast, uterine, liver, gastric, and colorectal cancers;carbohydrate antigen 15-3 (CA15-3), e.g., for lung, pancreatic, breast,ovarian, and liver cancers; carbohydrate antigen 19-9 (CA19-9), e.g.,for lung, ovarian, uterine, liver, gastric, colorectal, and bile ductcancers; cancer antigen 125 (CA125), e.g., for lung, pancreas, breast,ovarian, cervical, uterine, liver, gastric, and colorectal cancers; freeprostate specific antigen and prostate specific antigen-alpha(1) (PSA),for prostate cancer; free prostate specific antigen (PSAF), for prostateand colorectal cancers; prostate specificantigen-alpha(1)antichymotrypsin complex (PSAC), for prostate cancer;prostatic acid phosphatase (PAP), for prostate cancer; humanthyroglobulin (hTG), for thyroid cancer or Wilm's tumor; human chorionicgonadaotropin beta (hCGb), e.g., for lung, pancreatic, kidney, ovarian,uterine, testicular, liver, colorectal, bladder, and brain cancers;ferritin (Ferr), e.g., for lung cancer, testicular cancer, cancer of thelarynx, Burkitt's lymphoma, neuroblastoma, and leukemia; neuron specificenolase (NSE), for lung cancer, thyroid cancer, Wilm's tumor, andneuroblastoma; interleukin 2 (IL-2), for kidney cancer and multiplemyeloma; interleukin 6 (IL-6), for kidney cancer, breast cancer, ovariancancer, and multiple myeloma; beta 2 microglobulin (B2M), for kidneycancer, ovarian cancer, prostate cancer, leukemia, multiple myeloma, andlymphoma; and alpha 2 microglobulin (A2M), for prostate cancer. Theselection of biological sample (such as blood or urine) in which theaforementioned cancer markers are diagnostic and/or prognostic can bereadily determined by those skilled in the art.

As indicated above, the present invention provides a method formonitoring, diagnosing, or for the prognosis of cancer, such as ovariancancer, in a subject by detecting Bcl-2 in a biological sample from thesubject. In an embodiment, the method comprises contacting the samplewith an antibody specific for Bcl-2 which is directly or indirectlylabeled with a detectable substance, and detecting the detectablesubstance.

The methods of the invention may be used for the detection of either anover- or an under-abundance of Bcl-2 relative to a non-disorder state orthe presence of a modified (e.g., less than full length) Bcl-2 whichcorrelates with a disorder state (e.g., ovarian cancer), or aprogression toward a disorder state. The methods described herein may beused to evaluate the probability of the presence of malignant orpre-malignant cells. Such methods can be used to detect tumors,quantitate their growth, and assistg in the diagnosis and prognosis ofgynecological cancer. The methods can be used to detect the presence ofcancer metastasis, as well as confirm the absence or removal of alltumor tissue following surgery, cancer chemotherapy, and/or radiationtherapy. They can further be used to monitor cancer chemotherapy andtumor reappearance.

The methods of the invention are particularly useful in the diagnosis ofearly stage ovarian cancer (e.g., when the subject is asymptomatic) andfor the prognosis of ovarian cancer disease progression and mortality.As illustrated herein, increased levels of Bcl-2 detected in a sample(e.g., urine, serum, plasma, whole blood, ascites) compared to astandard (e.g., levels for normal or benign disorders) are indicative ofadvanced disease stage, serous histological type, suboptimal debulking,large residual tumor, and/or increased risk of disease progression andmortality.

The terms “sample”, “biological sample”, and the like refer to a type ofmaterial known to or suspected of expressing or containing Bcl-2, suchas urine. The test sample can be used directly as obtained from thesource or following a pretreatment to modify the character of thesample. The sample can be derived from any biological source, such astissues or extracts, including cells (e.g., tumor cells) andphysiological fluids, such as, for example, whole blood, plasma, serum,peritoneal fluid, ascites, and the like. The sample can be obtained fromanimals, preferably mammals, most preferably humans. The sample can bepretreated by any method and/or can be prepared in any convenient mediumthat does not interfere with the assay. The sample can be treated priorto use, such as preparing plasma from blood, diluting viscous fluids,applying one or more protease inhibitors to samples such as urine (e.g.,4-(2 aminoethyl)-benzene sulfonyl fluoride, EDTA, leupeptin, and/orpepstatin), and the like. Sample treatment can involve filtration,distillation, extraction, concentration, inactivation of interferingcomponents, the addition of reagents, and the like.

The presence of bcl-2 may be detected in a variety of biologicalsamples, including tissues or extracts thereof. Preferably, Bcl-2 isdetected in human urine.

In embodiments of the invention, the method described herein is adaptedfor diagnosing and monitoring gynecological cancer by quantitating Bcl-2in biological samples from a subject. Preferably, the amount of Bcl-2quantitated in a sample from a subject being tested is compared tolevels quantitated for another sample or an earlier sample from thesubject, or levels quantitated for a control sample. Levels for controlsamples from healthy subjects may be established by prospective and/orretrospective statistical studies. Healthy subjects who have noclinically evident disease or abnormalities may be selected forstatistical studies. Diagnosis may be made by a finding of statisticallydifferent levels of Bcl-2 compared to a control sample or previouslevels quantitated for the same subject.

The term “Bcl-2” refers to human B-cell lymphoma protein 2 (also knownas B-cell CLL/lymphoma 2), an integral outer mitochondrial protein thatblocks the apoptotic death of some cells such as lymphocytes (Cleary M.L. et al., Cell, 1986, 47(1):19-28; Tsujimoto Y. and Croce C. M., Proc.Natl. Acad. Sci. USA, 1986, 83:5214-5218, which are incorporated hereinby reference in their entirety). The term “Bcl-2” includes nucleic acidsequences (e.g., GenBank Accession No. M14745; SEQ ID NO:1) encoding theBcl-2 gene product (polypeptide), as well as the Bcl-2 polypeptide(e.g., GenBank Accession No. AAA35591; SEQ ID NO:2). The term includesall homologs, naturally occurring allelic variants, isoforms andprecursors of human Bcl-2 of GenBank Accession Nos. M14745 and AAA35591.In general, naturally occurring allelic variants of human Bcl-2 willshare significant sequence homology (70-90%) to the sequences shown inGenBank Accession Nos. M14745 and AAA35591. Allelic variants may containconservative amino acid substitutions from the Bcl-2 sequence or willcontain a substitution of an amino acid from a corresponding position ina Bcl-2 homologue. Two transcript variants, alpha and beta, produced byalternative splicing, differ in their C-terminal ends. The alpha variant(GenBank Accession No. NP_(—)000624 (SEQ ID NO:4); and GenBank AccessionNo. NM_(—)000633 (SEQ ID NO:3)) represents the longer transcript andencodes the longer isoform (alpha), and beta being the shorter (GenBankAccession No. NM_(—)000648 (SEQ ID NO:6); GenBank Accession No.NP_(—)000657 (SEQ ID NO:5). The beta variant differs in the 3′ UTR andcoding region compared to the alpha variant, as well as the C-terminalend. In a particular embodiment, the methods, devices, and kits of theinvention are specific for Bcl-2 (e.g., SEQ ID NOs: 1, 2, 3, 4, 5,and/or 6), but not nucleic acid molecules or polypeptides known in theart as “Bcl-2-like” molecules (e.g., employing binding agents specificfor (e.g., immunoreactive with) Bcl-2, but not reactive with Bcl-2 likemolecules), such as those described in Ruben et al., U.S. PatentApplication Publication 2002/0106731 A1, published Aug. 8, 2002, whichis incorporated herein by reference in its entirety.

The terms “subject” and “patient” are used interchangeably herein torefer to a warm-blooded animal, such as a mammal, which may be afflictedwith cancer. In some cancers, the subject is human or non-humanmammalian female. In other cancers, the subject is a human or non-humanmammalian male.

Agents that are capable of detecting Bcl-2 in the biological samples ofsubjects are those that interact or bind with the Bcl-2 polypeptide orthe nucleic acid molecule encoding Bcl-2. Examples of such agents (alsoreferred to herein as binding agents) include, but are not limited to,Bcl-2 antibodies or fragments thereof that bind Bcl-2, Bcl-2 bindingpartners, and nucleic acid molecules that hybridize to the nucleic acidmolecules encoding Bcl-2 polypeptides. Preferably, the binding agent islabeled with a detectable substance (e.g., a detectable moiety). Thebinding agent may itself function as a label.

Bcl-2 Antibodies

Antibodies specific for Bcl-2 that are used in the methods of theinvention may be obtained from scientific or commercial sources.Alternatively, isolated native Bcl-2 or recombinant Bcl-2 may beutilized to prepare antibodies, monoclonal or polyclonal antibodies, andimmunologically active fragments (e.g., a Fab or (Fab)₂ fragment), anantibody heavy chain, an antibody light chain, humanized antibodies, agenetically engineered single chain F, molecule (Ladne et al., U.S. Pat.No. 4,946,778), or a chimeric antibody, for example, an antibody whichcontains the binding specificity of a murine antibody, but in which theremaining portions are of human origin. Antibodies including monoclonaland polyclonal antibodies, fragments and chimeras, may be prepared usingmethods known to those skilled in the art. Preferably, antibodies usedin the methods of the invention are reactive against Bcl-2 if they bindwith a K_(a) of greater than or equal to 10⁷ M. In a sandwichimmunoassay of the invention, mouse polyclonal antibodies and rabbitpolyclonal antibodies are utilized.

In order to produce monoclonal antibodies, a host mammal is inoculatedwith a Bcl-2 protein or peptide and then boosted. Spleens are collectedfrom inoculated mammals a few days after the final boost. Cellsuspensions from the spleens are fused with a tumor cell in accordancewith the general method described by Kohler and Milstein (Nature, 1975,256:495-497). In order to be useful, a peptide fragment must containsufficient amino acid residues to define the epitope of the Bcl-2molecule being detected.

If the fragment is too short to be immunogenic, it may be conjugated toa carrier molecule. Some suitable carrier molecules include keyholelimpet hemocyanin and bovine serum albumin. Conjugation may be carriedout by methods known in the art. One such method is to combine acysteine residue of the fragment with a cysteine residue on the carriermolecule. The peptide fragments may be synthesized by methods known inthe art. Some suitable methods are described by Stuart and Young in“Solid Phase Peptide Synthesis,” Second Edition, Pierce Chemical Company(1984).

Purification of the antibodies or fragments can be accomplished by avariety of methods known to those of skill including, precipitation byammonium sulfate or sodium sulfate followed by dialysis against saline,ion exchange chromatography, affinity or immunoaffinity chromatographyas well as gel filtration, zone electrophoresis, etc. (Goding in,Monoclonal Antibodies: Principles and Practice, 2d ed., pp. 104-126,Orlando, Fla., Academic Press). It is preferable to use purifiedantibodies or purified fragments of the antibodies having at least aportion of a Bcl-2 binding region, including such as Fv, F(ab′)₂, Fabfragments (Harlow and Lane, 1988, Antibody Cold Spring Harbor) for thedetection of Bcl-2 in the fluids of gynecological cancer patients orthose at risk, preferably in the urine or blood of ovarian cancerpatients.

For use in detection and/or monitoring of cancer, the purifiedantibodies can be covalently attached, either directly or via linker, toa compound which serves as a reporter group to permit detection of thepresence of Bcl-2. A variety of different types of substances can serveas the reporter group, including but not limited to enzymes, dyes,radioactive metal and non-metal isotopes, fluorogenic compounds,fluorescent compounds, etc. Methods for preparation of antibodyconjugates of the antibodies (or fragments thereof) of the inventionuseful for detection, monitoring are described in U.S. Pat. Nos.4,671,958; 4,741,900 and 4,867,973.

In one aspect of the invention, preferred binding epitopes may beidentified from a known Bcl-2 gene sequence and its encoded amino acidsequence and used to generate Bcl-2 antibodies with high bindingaffinity. Also, identification of binding epitopes on Bcl-2 can be usedin the design and construction of preferred antibodies. For example, aDNA encoding a preferred epitope on Bcl-2 may be recombinantly expressedand used to select an antibody which binds selectively to that epitope.The selected antibodies then are exposed to the sample under conditionssufficient to allow specific binding of the antibody to the specificbinding epitope on Bcl-2 and the amount of complex formed then detected.Specific antibody methodologies are well understood and described in theliterature. A more detailed description of their preparation can befound, for example, in Practical Immunology, Butt, W. R., ed., MarcelDekker, New York, 1984.

The present invention also contemplates the detection of Bcl-2antibodies. Bcl-2 is a gynecological cancer-specific marker. Thus,detection of Bcl-2 antibodies in biological fluids of a subject mayenable the diagnosis of gynecological cancer.

Protein Binding Assays

Antibodies specifically reactive with Bcl-2, or derivatives, such asenzyme conjugates or labeled derivatives, may be used to detect Bcl-2 invarious biological samples, for example they may be used in any knownimmunoassays which rely on the binding interaction between an antigenicdeterminant of a protein and the antibodies. Examples of such assays areradioimmunoassays, enzyme immunoassay (e.g., ELISA), immunofluorescence,immnunoprecipitation, latex agglutination, hemagglutination, andhistochemical tests.

An antibody specific for Bcl-2 can be labeled with a detectablesubstance and localized in biological samples based upon the presence ofthe detectable substance. Examples of detectable substances include, butare not limited to, the following radioisotopes (e.g., ³H, ¹⁴C, ³⁵S,¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), luminescent labels such as luminol; enzymatic labels (e.g.,horseradish peroxidase, beta-galactosidase, luciferase, alkallinephosphatase, acetylcholinestease), biotinyl groups (which can bedetected by marked avidin, e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods), predetermined polypeptide epitopes recognized bya secondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). Indirectmethods may also be employed in which the primary antigen-antibodyreaction is amplified by the introduction of a second antibody, havingspecificity for the antibody reactive against Bcl-2. By way of example,if the antibody having specificity against Bcl-2 is a rabbit IgGantibody, the second antibody may be goat anti-rabbit gamma-globulinlabeled with a detectable substance as described herein.

Methods for conjugating or labeling the antibodies discussed above maybe readily accomplished by one of ordinary skill in the art. (See, forexample, Imman, Methods In Enzymology, Vol. 34, Affinity Techniques,Enzyme Purification: Part B, Jakoby and Wichek (eds.), Academic Press,New York, p. 30, 1974; and Wilchek and Bayer, “The Avidin-Biotin Complexin Bioanalytical Applications,” Anal. Biochem. 171:1-32, 1988, regardingmethods for conjugating or labeling the antibodies with an enzyme orligand binding partner).

Time-resolved fluorometry may be used to detect a signal. For example,the method described in Christopoulos T. K. and Diamandis E. P., Anal.Chem., 1992:64:342-346 may be used with a conventional time-resolvedfluorometer.

Therefore, in accordance with an embodiment of the invention, a methodis provided wherein a Bcl-2 antibody is labeled with an enzyme, asubstrate for the enzyme is added wherein the substrate is selected sothat the substrate, or a reaction product of the enzyme and substrate,forms fluorescent complexes with a lanthanide metal. A lanthanide metalis added and Bcl-2 is quantitated in the sample by measuringfluorescence of the fluorescent complexes. The antibodies specific forBcl-2 may be directly or indirectly labeled with an enzyme. Enzymes areselected based on the ability of a substrate of the enzyme, or areaction product of the enzyme and substrate, to complex with lanthanidemetals such as europium and terbium. Examples of suitable enzymesinclude alkalline phosphatase and beta-galactosidase. Preferably, theenzyme is akline phosphatase. The Bcl-2 antibodies may also beindirectly labeled with an enzyme. For example, the antibodies may beconjugated to one partner of a ligand binding pair, and the enzyme maybe coupled to the other partner of the ligand binding pair.Representative examples include avidin-biotin, and riboflavin-riboflavinbinding protein. Preferably the antibodies are biotinylated, and theenzyme is coupled to streptavidin.

In an embodiment of the method, antibody bound to Bcl-2 in a sample isdetected by adding a substrate for the enzyme. The substrate is selectedso that in the presence of a lanthanide metal (e.g., europium, terbium,samarium, and dysprosium, preferably europium and terbium), thesubstrate or a reaction product of the enzyme and substrate, forms afluorescent complex with the lanthanide metal. Examples of enzymes andsubstrates for enzymes that provide such fluorescent complexes aredescribed in U.S. Pat. No. 5,3112,922 to Diamandis. By way of example,when the antibody is directly or indirectly labeled with alkallinephosphatase, the substrate employed in the method may be4-methylumbeliferyl phosphate, or 5-fluorpsalicyl phosphate. Thefluorescence intensity of the complexes is typically measured using atime-resolved fluorometer, e.g., a CyberFluor 615 Immoanalyzer (NordionInternational, Kanata Ontario).

The sample, antibody specific for Bcl-2, or Bcl-2, may be immobilized ona carrier. Examples of suitable carriers are agarose, cellulose,dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulosepolystyrene, filter paper, ion-exchange resin, plastic film, plastictube, glass beads, polyamine-methyl vinyl ether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.The carrier may be in the shape of, for example, a tube, test plate,well, beads, disc, sphere, etc. The immobilized antibody may be preparedby reacting the material with a suitable insoluble carrier using knownchemical or physical methods, for example, cyanogen bromide coupling.

In accordance with an embodiment, the present invention provides a modefor determining Bcl-2 in an appropriate sample such as urine bymeasuring Bcl-2 by immunoassay. It will be evident to a skilled artisanthat a variety of immunoassay methods can be used to measure Bcl-2. Ingeneral, a Bcl-2 immunoassay method may be competitive orrioncompetitive. Competitive methods typically employ an immobilized orimmobilizable antibody to Bcl-2 (anti-Bcl-2) and a labeled form ofBcl-2. Sample Bcl-2 and labeled Bcl-2 compete for binding to anti-Bcl-2.After separation of the resulting labeled Bcl-2 that has become bound toanti-Bcl-2 (bound fraction) from that which has remained unbound(unbound fraction), the amount of the label in either bound or unboundfraction is measured and may be correlated with the amount of Bcl-2 inthe biological sample in any conventional manner, e.g., by comparison toa standard curve.

Preferably, a noncompetitive method is used for the determination ofBcl-2, with the most common method being the “sandwich” method. In thisassay, two anti-Bcl-2 antibodies are employed. One of the anti-Bcl-2antibodies is directly or indirectly labeled (also referred to as the“detection antibody”) and the other is immobilized or immobilizable(also referred to as the “capture antibody”). The capture and detectionantibodies can be contacted simultaneously or sequentially with thebiological sample. Sequential methods can be accomplished by incubatingthe capture antibody with the sample, and adding the detection antibodyat a predetermined time thereafter (sometimes referred to as the“forward” method); or the detection antibody can be incubated with thesample first and then the capture antibody added (sometimes referred toas the “reverse” method). After the necessary incubation(s) haveoccurred, to complete the assay, the capture antibody is separated fromthe liquid test mixture, and the label is measured in at least a portionof the separated capture antibody phase or the remainder of the liquidtest mixture. Generally, it is measured in the capture antibody phasesince it comprises Bcl-2 bound by (“sandwiched” between) the capture anddetection antibodies.

In a typical two-site immunometric assay for Bcl-2, one or both of thecapture and detection antibodies are polyclonal antibodies. The labelused in the detection antibody can be selected from any of those knownconventionally in the art. As with other embodiments of the proteindetection assay, the label can be an enzyme or a chemiluminescentmoiety, for example, or a radioactive isotope, a fluorophore, adetectable ligand (e.g., detectable by a secondary binding by a labeledbinding partner for the ligand), and the like. Preferably, the antibodyis labeled with an enzyme that is detected by adding a substrate that isselected so that a reaction product of the enzyme and substrate formsfluorescent complexes. The capture antibody is selected so that itprovides a mode for being separated from the remainder of the testmixture. Accordingly, the capture antibody can be introduced to theassay in an already immobilized or insoluble form, or can be in animmobilizable form, that is, a form which enables immobilization to beaccomplished subsequent to introduction of the capture antibody to theassay. An immobilized capture antibody can comprise an antibodycovalently or noncovalently attached to a solid phase such as a magneticparticle, a latex particle, a microtiter multi-well plate, a bead, acuvette, or other reaction vessel. An example of an immobilizablecapture antibody is an antibody that has been chemically modified with aligand moiety, e.g., a hapten, biotin, or the like, and that can besubsequently immobilized by contact with an immobilized form of abinding partner for the ligand, e.g., an antibody, avidin, or the like.In an embodiment, the capture antibody can be immobilized using aspecies specific antibody for the capture antibody that is bound to thesolid phase.

A particular sandwich immunoassay method of the invention employs twoantibodies reactive against Bcl-2, a second antibody having specificityagainst an antibody reactive against Bcl-2 labeled with an enzymaticlabel, and a fluorogenic substrate for the enzyme. In an embodiment, theenzyme is alkalline phosphatase (ALP) and the substrate is5-fluorosalicyl phosphate. ALP cleaves phosphate out of the fluorogenicsubstrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid(FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternarycomplex of the form FSA-Tb(3+)-EDTA, which can be quantified bymeasuring the Tb³⁺ fluorescence in a time-resolved mode. Fluorescenceintensity is typically measured using a time-resolved fluorometry asdescribed herein.

The above-described immunoassay methods and formats are intended to beexemplary and are not limiting since, in general, it will be understoodthat any immunoassay method or format can be used in the presentinvention.

The protein detection methods, devices, and kits of the invention canutilize nanowire sensor technology (Zhen et al., Nature Biotechnology,2005, 23(10):1294-1301; Lieber et al., Anal. Chem., 2006,78(13):4260-4269, which are incorporated herein by reference) ormicrocantilever technology (Lee et al., Biosens. Bioelectron, 2005,20(10):2157-2162; Wee et al., Biosens. Bioelectron., 2005,20(10):1932-1938; Campbell and Mutharasan, Biosens. Bioelectron., 2005,21(3):462-473; Campbell and Mutharasan, Biosens. Bioelectron., 2005,21(4):597-607; Hwang et al., Lab Chip, 2004, 4(6):547-552; Mukhopadhyayet al., Nano. Lett., 2005, 5(12):2835-2388, which are incorporatedherein by reference) for detection of Bcl-2 in samples. In addition,Huang et al. describe a prostate specific antigen immunoassay on acommercially available surface plasmon resonance biosensor (Biosens.Bioelectron., 2005, 21(3):483-490, which is incorporated herein byreference) which may be adapted for detection of Bcl-2. High-sensitivityminiaturized immunoassays may also be utilized for detection of Bcl-2(Cesaro-Tadic et al, Lab Chip, 2004, 4(6):563-569; Zimmerman et al.,Biomed. Microdevices, 2005, 7(2):99-110, which are incorporated hereinby reference).

Nucleic Acids

Nucleic acids including naturally occurring nucleic acids,oligonucleotides, antisense oligonucleotides, and syntheticoligonucleotides that hybridize to the nucleic acid encoding Bcl-2, areuseful as agents to detect the presence of Bcl-2 in the biologicalsamples of gynecological cancer patients or those at risk ofgynecological cancer, preferably in the urine of ovarian cancer patientsor those at risk of ovarian cancer. The present invention contemplatesthe use of nucleic acid sequences corresponding to the coding sequenceof Bcl-2 and to the complementary sequence thereof, as well as sequencescomplementary to the Bcl-2 transcript sequences occurring furtherupstream or downstream from the coding sequence (e.g., sequencescontained in, or extending into, the 5′ and 3′ untranslated regions) foruse as agents for detecting the expression of Bcl-2 in biologicalsamples of gynecological cancer patients, or those at risk ofgynecological cancer, preferably in the urine of ovarian cancer patientsor those at risk of ovarian cancer.

The preferred oligonucleotides for detecting the presence of Bcl-2 inbiological samples are those that are complementary to at least part ofthe cDNA sequence encoding Bcl-2. These complementary sequences are alsoknown in the art as “antisense” sequences. These oligonucleotides may beoligoribonucleotides or oligodeoxyribonucleotides. In addition,oligonucleotides may be natural oligomers composed of the biologicallysignificant nucleotides, i.e., A (adenine), dA (deoxyadenine), G(guanine), dG (deoxyguanine), C (cytosine), dC (deoxycytosine), T(thymine) and U (uracil), or modified oligonucleotide species,substituting, for example, a methyl group or a sulfur atom for aphosphate oxygen in the inter-nucleotide phosohodiester linkage.Additionally, these nucleotides themselves, and/or the ribose moietiesmay be modified.

The oligonucleotides may be synthesized chemically, using any of theknown chemical oligonucleotide synthesis methods well described in theart. For example, the oligonucleotides can be prepared by using any ofthe commercially available, automated nucleic acid synthesizers.Alternatively, the oligonucleotides may be created by standardrecombinant DNA techniques, for example, inducing transcription of thenoncoding strand. The DNA sequence encoding Bcl-2 may be inverted in arecombinant DNA system, e.g., inserted in reverse orientation downstreamof a suitable promoter, such that the noncoding strand now istranscribed.

Although any length oligonucleotide may be utilized to hybridize to anucleic acid encoding Bcl-2, oligonucleotides typically within the rangeof 8-100 nucleotides are preferred. Most preferable oligonucleotides foruse in detecting Bcl-2 in urine samples are those within the range of15-50 nucleotides.

The oligonucleotide selected for hybridizing to the Bcl-2 nucleic acidmolecule, whether synthesized chemically or by recombinant DNAtechnology, is then isolated and purified using standard techniques andthen preferably labeled (e.g., with ³⁵S or ³²P) using standard labelingprotocols.

The present invention also contemplates the use of oligonucleotide pairsin polymerize chain reactions (PCR) to detect the expression of Bcl-2 inbiological samples. The oligonucleotide pairs include a forward Bcl-2primer and a reverse Bcl-2 primer.

The presence of Bcl-2 in a sample from a patient may be determined bynucleic acid hybridization, such as but not limited to Northern blotanalysis, dot blotting, Southern blot analysis, fluorescence in situhybridization (FISH), and PCR. Chromatography, preferably HPLC, andother known assays may also be used to determine messenger RNA levels ofBcl-2 in a sample.

The Bcl-2 encoding nucleic acid molecules conceivably may be found inthe biological fluids inside a Bcl-positive cancer cell that is beingshed or released in the fluid under investigation.

In one aspect, the present invention contemplates the use of nucleicacids as agents for detecting Bcl-2 in biological samples of patients,wherein the nucleic acids are labeled. The nucleic agents may be labeledwith a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a calorimetric tag or other labels or tags thatare discussed above or that are known in the art.

In another aspect, the present invention contemplates the use ofNorthern blot analysis to detect the presence of Bcl-2 mRNA in a sampleof bodily fluid. The first step of the analysis involves separating asample containing Bcl-2 nucleic acid by gel electrophoresis. Thedispersed nucleic acids are then transferred to a nitrocellulose filteror another filter. Subsequently, the labeled oligonucleotide is exposedto the filter under suitable hybridizing conditions, e.g., 50%formamide, 5×SSPE, 2×Denhardt's solution, 0.1% SDS at 42° C., asdescribed in Molecular Cloning: A Laboratory Manual, Maniatis et al.(1982, CSH Laboratory). Other useful procedures known in the art includesolution hybridization, dot and slot RNA hybridization, and probe basedmicroarrays. Measuring the radioactivity of hybridized fragments, usingstandard procedures known in the art quantitates the amount of Bcl-2nucleic acid present in the biological fluid of a patient.

Dot blotting involves applying samples containing the nucleic acid ofinterest to a membrane. The nucleic acid can be denatured before orafter application to the membrane. The membrane is incubated with alabeled probe. Dot blot procedures are well known to the skilled artisanand are described more fully in U.S. Pat. Nos. 4,582,789 and 4,617,261,the disclosures of which are incorporated herein by reference.

Polymerase chain reaction (PCR) is a process for amplifying one or morespecific nucleic acid sequences present in a nucleic acid sample usingprimers and agents for polymerization and then detecting the amplifiedsequence. The extension product of one primer when hybridized to theother becomes a template for the production of the desired specificnucleic acid sequence, and vice versa, and the process is repeated asoften as is necessary to produce the desired-amount of the sequence. Theskilled artisan to detect the presence of desired sequence (U.S. Pat.No. 4,683,195) routinely uses polymerase chain reaction.

A specific example of PCR that is routinely performed by the skilledartisan to detect desired sequences is reverse transcript PCR (RT-PCR;Saiki et al., Science, 1985, 230:1350; Scharf et al., Science, 1986,233:1076). RT-PCR involves isolating total RNA from biological fluid,denaturing the RNA in the presence of primers that recognize the desirednucleic acid sequence, using the primers to generate a cDNA copy of theRNA by reverse transcription, amplifying the cDNA by PCR using specificprimers, and detecting the amplified cDNA by electrophoresis or othermethods known to the skilled artisan.

In a preferred embodiment, the methods of detecting Bcl-2 nucleic acidin biological fluids of gynecological cancer patients or those at riskthereof, preferably urine of ovarian cancer patients or those at riskthereof, include Northern blot analysis, dot blotting, Southern blotanalysis, FISH, and PCR.

Devices

The methods of the invention can be carried out on a solid support. Thesolid supports used may be those which are conventional for the purposeof assaying an analyte in a biological sample, and are typicallyconstructed of materials such as cellulose, polysaccharide such asSephadex, and the like, and may be partially surrounded by a housing forprotection and/or handling of the solid support. The solid support canbe rigid, semi-rigid, flexible, elastic (having shape-memory), etc.,depending upon the desired application. Bcl-2 can be detected in asample in vivo or in vitro (ex vivo). When, according to an embodimentof the invention, the amount of Bcl-2 in a sample is to be determinedwithout removing the sample from the body (i.e., in vivo), the supportshould be one which is harmless to the subject and may be in any formconvenient for insertion into an appropriate part of the body. Forexample, the support may be a probe made of polytetrafluoroethylene,polystyrene or other rigid non-harmful plastic material and having asize and shape to enable it to be introduced into a subject. Theselection of an appropriate inert support is within the competence ofthose skilled in the art, as are its dimensions for the intendedpurpose.

A contacting step in the assay (method) of the invention can involvecontacting, combining, or mixing the biological sample and the solidsupport, such as a reaction vessel, microvessel, tube, microtube, well,multi-well plate, or other solid support. In an embodiment of theinvention, the solid support to be contacted with the biological sample(e.g., urine) has an absorbent pad or membrane for lateral flow of theliquid medium to be assayed, such as those available from MilliporeCorp. (Bedford, Mass.), including but not limited to Hi-Flow Plus™membranes and membrane cards, and SureWick™ pad materials.

The diagnostic device useful in carrying out the methods of theinvention can be constructed in any form adapted for the intended use.Thus, in one embodiment, the device of the invention can be constructedas a disposable or reusable test strip or stick to be contacted with abiological sample such as urine or blood for which Bcl-2 level is to bedetermined. In another embodiment, the device can be constructed usingart recognized micro-scale manufacturing techniques to produceneedle-like embodiments capable of being implanted or injected into ananatomical site, such as the peritoneal cavity, for indwellingdiagnostic applications. In other embodiments, devices intended forrepeated laboratory use can be constructed in the form of an elongatedprobe.

In preferred embodiments, the devices of the invention comprise a solidsupport (such as a strip or dipstick), with a surface that functions asa lateral flow matrix defining a flow path for a biological sample suchas urine, whole blood, serum, plasma, peritoneal fluid, or ascites.

Immunochromatographic assays, also known as lateral flow test strips orsimply strip tests, for detecting various analytes of interest, havebeen known for some time, and may be used for detection of Bcl-2. Thebenefits of lateral flow tests include a user-friendly format, rapidresults, long-term stability over a wide range of climates, andrelatively low cost to manufacture. These features make lateral flowtests ideal for applications involving home testing, rapid point of caretesting, and testing in the field for various analytes. The principlebehind the test is straightforward. Essentially, any ligand that can bebound to a visually detectable solid support, such as dyed microspheres,can be tested for, qualitatively, and in many cases evensemi-quantitatively. For example, a one-step lateral flow immunostripfor the detection of free and total prostate specific antigen in serumis described in Fernandez-Sanchez et al. (J. Immuno. Methods, 2005,307(1-2):1-12, which is incorporated herein by reference) and may beadapted for detection of Bcl-2 in a biological sample such as blood orurine.

Some of the more common immunochromatographic assays currently on themarket are tests for pregnancy (as an over-the-counter (OTC) test kit),Strep throat, and Chlamydia. Many new tests for well-known antigens havebeen recently developed using the immunochromatographic assay method.For instance, the antigen for the most common cause of communityacquired pneumonia has been known since 1917, but a simple assay wasdeveloped only recently, and this was done using this simple test stripmethod (Murdoch, D. R. et al. J Clin Microbiol, 2001, 39:3495-3498).Human immunodeficiency virus (HIV) has been detected rapidly in pooledblood using a similar assay (Soroka, S. D. et al. J Clin Virol, 2003,27:90-96). A nitrocellulose membrane card has also been used to diagnoseschistosomiasis by detecting the movement and binding of nanoparticlesof carbon (van Dam, G. J. et al. J Clin Microbiol, 2004, 42:5458-5461).

The two common approaches to the immunochromatographic assay are thenon-competitive (or direct) and competitive (or competitive inhibition)reaction schemes (TechNote #303, Rev. #001, 1999, Bangs Laboratories,Inc., Fishers, Ind.). The direct (double antibody sandwich) format istypically used when testing for larger analytes with multiple antigenicsites such as luteinizing hormone (LH), human chorionic gonadotropin(hCG), and HIV. In this instance, less than an excess of sample analyteis desired, so that some of the microspheres will not be captured at thecapture line, and will continue to flow toward the second line ofimmobilized antibodies, the control zone. This control line usesspecies-specific anti-immunoglobulin antibodies, specific for theconjugate antibodies on the microspheres. Free antigen, if present, isintroduced onto the device by adding sample (urine, serum, etc.) onto asample addition pad. Free antigen then binds to antibody-microspherecomplexes. Antibody 1, specific for epitope 1 of sample antigen, iscoupled to dye microspheres and dried onto the device. When sample isadded, microsphere-antibody complex is rehydrated and carried to acapture zone and control lines by liquid. Antibody 2, specific for asecond antigenic site (epitope 2) of sample antigen, is dried onto amembrane at the capture line. Antibody 3, a species-specific,anti-immunoglobulin antibody that will react with antibody 1, is driedonto the membrane at the control line. If antigen is present in thesample (i.e., a positive test), it will bind by its two antigenic sites,to both antibody 1 (conjugated to microspheres) and antibody 2 (driedonto membrane at the capture line). Antibody 1-coated microspheres arebound by antibody 3 at the control line, whether antigen is present ornot. If antigen is not present in the sample (a negative test),microspheres pass the capture line without being trapped, but are caughtby the control line.

The competitive reaction scheme is typically used when testing for smallmolecules with single antigenic determinants, which cannot bond to twoantibodies simultaneously. As with double antibody sandwich assay, freeantigen, if present is introduced onto the device by adding sample ontoa sample pad. Free antigen present in the sample binds to anantibody-microsphere complex. Antibody 1 is specific for sample antigenand couple to dyed microspheres. An antigen-carrier molecule (typicallyBSA) conjugate is dried onto a membrane at the capture line. Antibody 2(Ab2) is dried onto the membrane at the control line, and is aspecies-specific anti-immunoglobulin that will capture the reagentparticles and confirm that the test is complete. If antigen is presentin the sample (a positive test), antibody on microspheres (Ab1) isalready saturated with antigen from sample and, therefore, antigenconjugate bound at the capture line does not bind to it. Anymicrospheres not caught by the antigen carrier molecule can be caught byAb2 on the control line. If antigen is not present in the sample (anegative test), antibody-coated dyed microspheres are allowed to becaptured by antigen conjugate bound at the capture line.

Normally, the membranes used to hold the antibodies in place on thesedevices are made of primary hydrophobic materials, such asnitrocellulose. Both the microspheres used as the solid phase supportsand the conjugate antibodies are hydrophobic, and their interaction withthe membrane allows them to be effectively dried onto the membrane.

Samples and/or Bcl-2-specific binding agents may be arrayed on the solidsupport, or multiple supports can be utilized, for multiplex detectionor analysis. “Arraying” refers to the act of organizing or arrangingmembers of a library (e.g., an array of different samples or an array ofdevices that target the same target molecules or different targetmolecules), or other collection, into a logical or physical array. Thus,an “array” refers to a physical or logical arrangement of, e.g.,biological samples. A physical array can be any “spatial format” orphysically gridded format” in which physical manifestations ofcorresponding library members are arranged in an ordered manner, lendingitself to combinatorial screening. For example, samples corresponding toindividual or pooled members of a sample library can be arranged in aseries of numbered rows and columns, e.g., on a multi-well plate.Similarly, binding agents can be plated or otherwise deposited inmicrotitered, e.g., 96-well, 384-well, or -1536 well, plates (or trays).Optionally, Bcl-2-specific binding agents may be immobilized on thesolid support.

Detection of Bcl-2 and cancer biomarkers, and other assays that are tobe carried out on samples, can be carried out simultaneously orsequentially with the detection of other target molecules, and may becarried out in an automated fashion, in a high-throughput format.

The Bcl-2-specific binding agents can be deposited but “free”(non-immobilized) in the conjugate zone, and be immobilized in thecapture zone of a solid support. The Bcl-2-specific binding agents maybe immobilized by non-specific adsorption onto the support or bycovalent bonding to the support, for example. Techniques forimmobilizing binding agents on supports are known in the art and aredescribed for example in U.S. Pat. Nos. 4,399,217, 4,381,291, 4,357,311,4,343,312 and 4,260,678, which are incorporated herein by reference.Such techniques can be used to immobilize the binding agents in theinvention. When the solid support is polytetrafluoroethylene, it ispossible to couple hormone antibodies onto the support by activating thesupport using sodium and ammonia to aminate it and covalently bondingthe antibody to the activated support by means of a carbodiimidereaction (yon Klitzing, Schultek, Strasburger, Fricke and Wood in“Radioimmunoassay and Related Procedures in Medicine 1982”,International Atomic Energy Agency, Vienna (1982), pages 57-62.).

The diagnostic device of the invention can utilize lateral flow strip(LFS) technology, which has been applied to a number of other rapidstrip assay systems, such as over-the-counter early pregnancy teststrips based on antibodies to human chorionic gonadotropin (hCG). Aswith many other diagnostic devices, the device utilizes a binding agentto bind the target molecule (Bcl-2). The device has an application zonefor receiving a biological sample such as blood or urine, a labelingzone containing label which binds to Bcl-2 in the sample, and adetection zone where Bcl-2 label is retained.

Binding agent retained in the detection zone gives a signal, and thesignal differs depending on whether Bcl-2 levels in the biologicalsample are lower than, equal to, or greater than a given thresholdconcentration. For example, in the case of urinary Bcl-2 for thedetection of ovarian cancer, the threshold concentration may be between0 ng/ml and 2.0 ng/ml. In another embodiment, in the case of urinaryBcl-2 for the detection of ovarian cancer, the threshold concentrationis 1.8 ng/ml. A sample from a subject having a Bcl-2 level equal to orgreater than the given reference Bcl-2 concentration can be referred toas a “threshold level”, “threshold amount”, or “threshold sample”. Theapplication zone in the device is suitable for receiving the biologicalsample to be assayed. It is typically formed from absorbent materialsuch as blotting paper. The labeling zone contains binding agent thatbinds to any Bcl-2 in the sample. In one embodiment, the binding agentis an antibody (e.g., monoclonal antibody, polyclonal antibody, antibodyfragment). For ease of detection, the binding agent is preferably inassociation with a label that provides a signal that is visible to thenaked eye, e.g., it is tagged with a fluorescent tag or a colored tagsuch as conjugated colloidal gold, which is visible as a pink color.

The detection zone retains Bcl-2 to which the binding agent has bound.This will typically be achieved using an immobilized binding agent suchas an immobilized antibody. Where the binding agent in the labeling zoneand the detection zone are both antibodies, they will typicallyrecognize different epitopes on the target molecule (Bcl-2 protein).This allows the formation of a “sandwich” comprisingantibody-Bcl-2-antibody.

The detection zone is downstream of the application zone, with thelabelling zone typically located between the two. A sample will thusmigrate from the application zone into the labeling zone, where any inthe sample binds to the label. Bcl-2-binding agent-complexes continue tomigrate into the detection zone together with excess binding agent. Whenthe Bcl-2-binding agent complex encounters the capture reagent, thecomplex is retained whilst the sample and excess binding agent continueto migrate. As Bcl-2 levels in the sample increase, the amount ofbinding agent (in the form of Bcl-2-binding agent complex) retained inthe detection zone increases proportionally.

In preferred embodiments, the device of the invention has the ability todistinguish between samples according to the threshold concentration.This can be achieved in various ways.

One type of device includes a reference zone that includes a signal offixed intensity against which the amount of binding agent retained inthe detection zone can be compared—when the signal in the detection zoneequals the signal in the reference zone, the sample is a thresholdsample; when the signal in the detection zone is less intense than thereference zone, the sample contains less Bcl-2 than a threshold sample;when the signal in the detection zone is more intense than the referencezone, the sample contains more Bcl-2 than a threshold sample.

A suitable reference zone can be prepared and calibrated withoutdifficulty. For this type of device, the binding agent will generally bepresent in excess to Bcl-2 in the sample, and the reference zone may beupstream or, preferably, downstream of the detection zone. The signal inthe reference zone will be of the same type as the signal in thedetection zone, i.e., they will typically both be visible to the nakedeye, e.g., they will use the same tag. A preferred reference zone in adevice of this type comprises immobilized protein (e.g., bovine serumalbumin) which is tagged with colloidal gold.

In another device of the invention, the reference zone is downstream ofthe detection zone and includes a reagent which captures binding agent(e.g., an immobilised anti-binding agent antibody). Binding agent thatflows through the device is not present in excess, but is at aconcentration such that 50% of it is bound by a sample having Bcl-2 atthe threshold concentration. In a threshold sample, therefore, 50% ofthe binding agent will be retained in the detection zone and 50% in thereference zone. If the Bcl-2 level in the sample is greater than in athreshold sample, less than 50% of the binding agent will reach thereference zone and the detection zone will give a more intense signalthan the reference zone; conversely, if the Bcl-2 level in the sample isless than in a threshold sample, less than 50% of the binding agent willbe retained in the detection zone and the reference zone will give amore intense signal than the detection zone.

In another device of the invention which operates according to similarprinciples, the reference zone is downstream of the detection zone andincludes a limiting amount of a reagent which captures binding agent(e.g., an immobilised anti-binding agent antibody). The reagent ispresent at a level such that it retains the same amount of label whichwould bind to the detection zone for a threshold sample, with excesslabel continuing to migrate beyond the reference zone.

In these three types of device, therefore, a comparison between thedetection zone and the reference zone is used to compare the sample withthe threshold concentration. The detection:reference binding ratio canpreferably be determined by eye. Close juxtaposition of the detectionand reference zones is preferred in order to facilitate visualcomparison of the signal intensities in the two zones.

In a fourth type of device, no reference zone is needed, but thedetection zone is configured such that it gives an essentially on/offresponse, e.g., no signal is given below the threshold concentrationbut, at or above the threshold, signal is given.

In a fifth type of device, no reference zone is needed, but an externalreference is used which corresponds to the threshold concentration. Thiscan take various forms, e.g., a printed card against which the signal inthe detection zone can be compared, or a machine reader which comparesan absolute value measured in the detection zone (e.g., a calorimetricsignal) against a reference value stored in the machine.

In some embodiments of the invention, the device includes a control zonedownstream of the detection zone. This will generally be used to captureexcess binding agent that passes through the detection and/or referencezones (e.g., using immobilized anti-binding agent antibody). Whenbinding agent is retained at the control zone, this confirms thatmobilization of the binding agent and migration through the device haveboth occurred. It will be appreciated that this function may be achievedby the reference zone.

In a preferred embodiment, the detection, reference and control zonesare preferably formed on a nitrocellulose support.

Migration from the application zone to the detection zone will generallybe assisted by a wick downstream of the detection zone to aid capillarymovement. This wick is typically formed from absorbent material such asblotting or chromatography paper.

The device of the invention can be produced simply and cheaply,conveniently in the form of a dipstick. Furthermore, it can be used veryeasily, for instance by the home user.

The invention thus provides a device which can be used at home as ascreen for cancer, such as ovarian cancer.

Kits for Diagnosing or Monitoring Gynecological Cancer

In one aspect, the present invention includes kits comprising therequired elements for diagnosing or monitoring cancer. Preferably, thekits comprise a container for collecting biological fluid from a patientand an agent for detecting the presence of Bcl-2 or its encoding nucleicacid in the fluid. The components of the kits can be packaged either inaqueous medium or in lyophilized form.

The methods of the invention can be carried out using a diagnostic kitfor qualitatively or quantitatively detecting Bcl-2 in a sample such asblood or urine. By way of example, the kit can contain binding agents(e.g., antibodies) specific for Bcl-2, antibodies against the antibodieslabeled with an enzyme; and a substrate for the enzyme. The kit can alsocontain a solid support such as microtiter multi-well plates, standards,assay diluent, wash buffer, adhesive plate covers, and/or instructionsfor carrying out a method of the invention using the kit. In oneembodiment, the kit includes one or protease inhibitors (e.g., aprotease inhibitor cocktail) to be applied to the biological sample tobe assayed (such as blood or urine).

Kits for diagnosing or monitoring gynecological cancer containing one ormore agents that detect the Bcl-2 protein, such as but not limited toBcl-2 antibodies, fragments thereof, or Bcl-2 binding partners, can beprepared. The agent(s) can be packaged with a container for collectingthe biological fluid from a patient. When the antibodies or bindingpartner are used in the kits in the form of conjugates in which a labelis attached, such as a radioactive metal ion or a moiety, the componentsof such conjugates can be supplied either in fully conjugated form, inthe form of intermediates or as separate moieties to be conjugated bythe user of the kit.

Kits containing one or more agents that detect Bcl-2 nucleic acid, suchas but not limited to the full length Bcl-2 nucleic acid, Bcl-2oligonucleotides, and pairs of Bcl-2 primers can also be prepared. Theagent(s) can be packaged with a container for collecting biologicalsamples from a patient. The nucleic acid can be in the labeled form orto be labeled form.

Other components of the kit may include but are not limited to, meansfor collecting biological samples, means for labeling the detectingagent (binding agent), membranes for immobilizing the Bcl-2 protein orBcl-2 nucleic acid in the biological sample, means for applying thebiological sample to a membrane, means for binding the agent to Bcl-2 inthe biological sample of a subject, a second antibody, a means forisolating total RNA from a biological fluid of a subject, means forperforming gel electrophoresis, means for generating cDNA from isolatedtotal RNA, means for performing hybridization assays, and means forperforming PCR, etc.

As used herein, the term “ELISA” includes an enzyme-linkedimmunoabsorbent assay that employs an antibody or antigen bound to asolid phase and an enzyme-antigen or enzyme-antibody conjugate to detectand quantify the amount of an antigen (e.g., Bcl-2) or antibody presentin a sample. A description of the ELISA technique is found in Chapter 22of the 4^(th) Edition of Basic and Clinical Immunology by D. P. Sites etal., 1982, published by Lange Medical Publications of Los Altos, Calif.and in U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043, thedisclosures of which are herein incorporated by reference. ELISA is anassay that can be used to quantitate the amount of antigen, proteins, orother molecules of interest in a sample. In particular, ELISA can becarried out by attaching on a solid support (e.g., polyvinylchloride) anantibody specific for an antigen or protein of interest. Cell extract orother sample of interest such as urine can be added for formation of anantibody-antigen complex, and the extra, unbound sample is washed away.An enzyme-linked antibody, specific for a different site on the antigenis added. The support is washed to remove the unbound enzyme-linkedsecond antibody. The enzyme-linked antibody can include, but is notlimited to, alkaline phosphatase. The enzyme on the second antibody canconvert an added colorless substrate into a colored product or canconvert a non-fluorescent substrate into a fluorescent product. TheELISA-based assay method provided herein can be conducted in a singlechamber or on an array of chambers and can be adapted for automatedprocesses.

In these exemplary embodiments, the antibodies can be labeled with pairsof FRET dyes, bioluminescence resonance energy transfer (BRET) protein,fluorescent dye-quencher dye combinations, beta gal complementationassays protein fragments. The antibodies may participate in FRET, BRET,fluorescence quenching or beta-gal complementation to generatefluorescence, calorimetric or enhanced chemiluminescence (ECL) signals,for example.

These methods are routinely employed in the detection ofantigen-specific antibody responses, and are well described in generalimmunology text books such as hnmunology by Ivan Roitt, JonathanBrostoff and David Male (London: Mosby, c1998. 5th ed. andImmunobiology: Immune System in Health and Disease/Charles A. Janewayand Paul Travers. Oxford: Blackwell Sci. Pub., 1994), the contents ofwhich are herein incorporated by reference.

Definitions

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth, i.e., proliferative disorders. Examples ofsuch proliferative disorders include cancers such as carcinoma,lymphoma, blastoma, sarcoma, and leukemia, as well as other cancersdisclosed herein. More particular examples of such cancers includebreast cancer, prostate cancer, colon cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, cervical cancer, ovarian cancer, peritonealcancer, liver cancer, e.g., hepatic carcinoma, bladder cancer,colorectal cancer, endometrial carcinoma, kidney cancer, and thyroidcancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); pancreatic cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas. Examples of cancertypes are listed in Table 1.

TABLE 1 Examples of Cancer Types Acute Lymphoblastic Leukemia, HairyCell Leukemia Adult Head and Neck Cancer Acute Lymphoblastic Leukemia,Hepatocellular (Liver) Cancer, Adult Childhood (Primary) Acute MyeloidLeukemia, Adult Hepatocellular (Liver) Cancer, Childhood Acute MyeloidLeukemia, Childhood (Primary) Adrenocortical Carcinoma Hodgkin'sLymphoma, Adult Adrenocortical Carcinoma, Hodgkin's Lymphoma, ChildhoodChildhood Hodgkin's Lymphoma During Pregnancy AIDS-Related CancersHypopharyngeal Cancer AIDS-Related Lymphoma Hypothalamic and VisualPathway Glioma, Anal Cancer Childhood Astrocytoma, Childhood CerebellarIntraocular Melanoma Astrocytoma, Childhood Cerebral Islet CellCarcinoma (Endocrine Pancreas) Basal Cell Carcinoma Kaposi's SarcomaBile Duct Cancer, Extrahepatic Kidney (Renal Cell) Cancer Bladder CancerKidney Cancer, Childhood Bladder Cancer, Childhood Laryngeal Cancer BoneCancer, Laryngeal Cancer, Childhood Osteosarcoma/Malignant FibrousLeukemia, Acute Lymphoblastic, Adult Histiocytoma Leukemia, AcuteLymphoblastic, Brain Stem Glioma, Childhood Childhood Brain Tumor, AdultLeukemia, Acute Myeloid, Adult Brain Tumor, Brain Stem Glioma, Leukemia,Acute Myeloid, Childhood Childhood Leukemia, Chronic Lymphocytic BrainTumor, Cerebellar Leukemia, Chronic Myelogenous Astrocytoma, ChildhoodLeukemia, Hairy Cell Brain Tumor, Cerebral Lip and Oral Cavity CancerAstrocytoma/Malignant Glioma, Liver Cancer, Adult (Primary) ChildhoodLiver Cancer, Childhood (Primary) Brain Tumor, Ependymoma, Lung Cancer,Non-Small Cell Childhood Lung Cancer, Small Cell Brain Tumor,Medulloblastoma, Lymphoma, AIDS-Related Childhood Lymphoma, Burkitt'sBrain Tumor, Supratentorial Lymphoma, Cutaneous T-Cell, see MycosisPrimitive Neuroectodermal Tumors, Fungoides and Sézary SyndromeChildhood Lymphoma, Hodgkin's, Adult Brain Tumor, Visual Pathway andLymphoma, Hodgkin's, Childhood Hypothalamic Glioma, Childhood Lymphoma,Hodgkin's During Pregnancy Brain Tumor, Childhood Lymphoma,Non-Hodgkin's, Adult Breast Cancer Lymphoma, Non-Hodgkin's, ChildhoodBreast Cancer, Childhood Lymphoma, Non-Hodgkin's During Breast Cancer,Male Pregnancy Bronchial Adenomas/Carcinoids, Lymphoma, Primary CentralNervous Childhood System Burkitt's Lymphoma Macroglobulinemia,Waldenström's Carcinoid Tumor, Childhood Malignant Fibrous Histiocytomaof Carcinoid Tumor, Gastrointestinal Bone/Osteosarcoma Carcinoma ofUnknown Primary Medulloblastoma, Childhood Central Nervous SystemLymphoma, Melanoma Primary Melanoma, Intraocular (Eye) CerebellarAstrocytoma, Childhood Merkel Cell Carcinoma CerebralAstrocytoma/Malignant Mesothelioma, Adult Malignant Glioma, ChildhoodMesothelioma, Childhood Cervical Cancer Metastatic Squamous Neck Cancerwith Childhood Cancers Occult Primary Chronic Lymphocytic LeukemiaMultiple Endocrine Neoplasia Syndrome, Chronic Myelogenous LeukemiaChildhood Chronic Myeloproliferative Disorders Multiple Myeloma/PlasmaCell Neoplasm Colon Cancer Mycosis Fungoides Colorectal Cancer,Childhood Myelodysplastic Syndromes Cutaneous T-Cell Lymphoma, seeMyelodysplastic/Myeloproliferative Mycosis Fungoides and Sézary DiseasesSyndrome Myelogenous Leukemia, Chronic Endometrial Cancer MyeloidLeukemia, Adult Acute Ependymoma, Childhood Myeloid Leukemia, ChildhoodAcute Esophageal Cancer Myeloma, Multiple Esophageal Cancer, ChildhoodMyeloproliferative Disorders, Chronic Ewing's Family of Tumors NasalCavity and Paranasal Sinus Cancer Extracranial Germ Cell Tumor,Nasopharyngeal Cancer Childhood Nasopharyngeal Cancer, ChildhoodExtragonadal Germ Cell Tumor Neuroblastoma Extrahepatic Bile Duct CancerNon-Hodgkin's Lymphoma, Adult Eye Cancer, Intraocular MelanomaNon-Hodgkin's Lymphoma, Childhood Eye Cancer, RetinoblastomaNon-Hodgkin's Lymphoma During Gallbladder Cancer Pregnancy Gastric(Stomach) Cancer Non-Small Cell Lung Cancer Gastric (Stomach) Cancer,Childhood Oral Cancer, Childhood Gastrointestinal Carcinoid Tumor OralCavity Cancer, Lip and Germ Cell Tumor, Extracranial, OropharyngealCancer Childhood Osteosarcoma/Malignant Fibrous Germ Cell Tumor,Extragonadal Histiocytoma of Bone Germ Cell Tumor, Ovarian OvarianCancer, Childhood Gestational Trophoblastic Tumor Ovarian EpithelialCancer Glioma, Adult Ovarian Germ Cell Tumor Glioma, Childhood BrainStem Ovarian Low Malignant Potential Tumor Glioma, Childhood CerebralPancreatic Cancer Astrocytoma Pancreatic Cancer, Childhood Glioma,Childhood Visual Pathway Pancreatic Cancer, Islet Cell and HypothalamicParanasal Sinus and Nasal Cavity Cancer Parathyroid Cancer Skin Cancer(Melanoma) Penile Cancer Skin Carcinoma, Merkel Cell PheochromocytomaSmall Cell Lung Cancer Pineoblastoma and Supratentorial Primitive SmallIntestine Cancer Neuroectodermal Tumors, Childhood Soft Tissue Sarcoma,Adult Pituitary Tumor Soft Tissue Sarcoma, Childhood Plasma CellNeoplasm/Multiple Myeloma Squamous Cell Carcinoma, see SkinPleuropulmonary Blastoma Cancer (non-Melanoma) Pregnancy and BreastCancer Squamous Neck Cancer with Occult Pregnancy and Hodgkin's LymphomaPrimary, Metastatic Pregnancy and Non-Hodgkin's Lymphoma Stomach(Gastric) Cancer Primary Central Nervous System Stomach (Gastric)Cancer, Childhood Lymphoma Supratentorial Primitive Prostate CancerNeuroectodermal Tumors, Childhood Rectal Cancer T-Cell Lymphoma,Cutaneous, see Renal Cell (Kidney) Cancer Mycosis Fungoides and SézaryRenal Cell (Kidney) Cancer, Childhood Syndrome Renal Pelvis and Ureter,Transitional Cell Testicular Cancer Cancer Thymoma, ChildhoodRetinoblastoma Thymoma and Thymic Carcinoma Rhabdomyosarcoma, ChildhoodThyroid Cancer Salivary Gland Cancer Thyroid Cancer, Childhood SalivaryGland Cancer, Childhood Transitional Cell Cancer of the Renal Sarcoma,Ewing's Family of Tumors Pelvis and Ureter Sarcoma, Kaposi'sTrophoblastic Tumor, Gestational Sarcoma, Soft Tissue, Adult UnknownPrimary Site, Carcinoma Sarcoma, Soft Tissue, Childhood of, AdultSarcoma, Uterine Unknown Primary Site, Cancer of, Sezary SyndromeChildhood Skin Cancer (non-Melanoma) Unusual Cancers of Childhood SkinCancer, Childhood Ureter and Renal Pelvis, Transitional Cell CancerUrethral Cancer Uterine Cancer, Endometrial Uterine Sarcoma VaginalCancer Visual Pathway and Hypothalamic Glioma, Childhood Vulvar CancerWaldenström's Macroglobulinemia Wilms' Tumor

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), ultrasound, CT, and MRI, or by needle aspirations. The useof these latter techniques is more common in early detection. Molecularand phenotypic analysis of cancer cells within a tissue will usuallyconfirm if the cancer is endogenous to the tissue or if the lesion isdue to metastasis from another site.

A “sample” (biological sample) can be any composition of matter ofinterest from a human or non-human subject, in any physical state (e.g.,solid, liquid, semi-solid, vapor) and of any complexity. The sample canbe any composition reasonably suspecting of containing Bcl-2 that can beanalyzed by the methods, devices, and kits of the invention. Preferably,the sample is a fluid (biological fluid). Samples can include human oranimal samples. The sample may be contained within a test tube, culturevessel, multi-well plate, or any other container or supportingsubstrate. The sample can be, for example, a cell culture, human oranimal tissue. Fluid homogenates of cellular tissues are biologicalfluids that may contain Bcl-2 for detection by the invention.

The “complexity” of a sample refers to the relative number of differentmolecular species that are present in the sample.

The terms “body fluid” and “bodily fluid”, as used herein, refer to acomposition obtained from a human or animal subject. Bodily fluidsinclude, but are not limited to, urine, whole blood, blood plasma,serum, tears, semen, saliva, sputum, exhaled breath, nasal secretions,pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations,interstitial fluid, lymph fluid, meningal fluid, amniotic fluid,glandular fluid, feces, perspiration, mucous, vaginal or urethralsecretion, cerebrospinal fluid, and transdermal exudate. Bodily fluidalso includes experimentally separated fractions of all of the precedingsolutions or mixtures containing homogenized solid material, such asfeces, tissues, and biopsy samples.

The term “ex vivo,” as used herein, refers to an environment outside ofa subject. Accordingly, a sample of bodily fluid collected from asubject is an ex vivo sample of bodily fluid as contemplated by thesubject invention. In-dwelling embodiments of the method and device ofthe invention obtain samples in vivo.

As used herein, the term “conjugate” refers to a compound comprising twoor more molecules bound together, optionally through a linking group, toform a single structure. The binding can be made by a direct connection(e.g., a chemical bond) between the molecules or by use of a linkinggroup.

As used herein, the terms solid “support”, “substrate”, and “surface”refer to a solid phase which is a porous or non-porous water insolublematerial that can have any of a number of shapes, such as strip, rod,particle, beads, or multi-welled plate. In some embodiments, the supporthas a fixed organizational support matrix that preferably functions asan organization matrix, such as a microtiter tray. Solid supportmaterials include, but are not limited to, cellulose, polysaccharidesuch as Sephadex, glass, polyacryloylmorpholide, silica, controlled poreglass (CPG), polystyrene, polystyrene/latex, polyethylene such as ultrahigh molecular weight polyethylene (UPE), polyamide, polyvinylidinefluoride (PVDF), polytetrafluoroethylene (PTFE; TEFLON), carboxylmodified teflon, nylon, nitrocellulose, and metals and alloys such asgold, platinum and palladium. The solid support can be biological,non-biological, organic, inorganic, or a combination of any of these,existing as particles, strands, precipitates, gels, sheets, pads, cards,strips, dipsticks, test strips, tubing, spheres, containers,capillaries, pads, slices, films, plates, slides, etc., depending uponthe particular application. Preferably, the solid support is planar inshape, to facilitate contact with a biological sample such as urine,whole blood, plasma, serum, peritoneal fluid, or ascites fluid. Othersuitable solid support materials will be readily apparent to those ofskill in the art. The solid support can be a membrane, with or without abacking (e.g., polystyrene or polyester card backing), such as thoseavailable from Millipore Corp. (Bedford, Mass.), e.g., Hi-Flow™ Plusmembrane cards. The surface of the solid support may contain reactivegroups, such as carboxyl, amino, hydroxyl, thiol, or the like for theattachment of nucleic acids, proteins, etc. Surfaces on the solidsupport will sometimes, though not always, be composed of the samematerial as the support. Thus, the surface can be composed of any of awide variety of materials, such as polymers, plastics, resins,polysaccharides, silica or silica-based materials, carbon, metals,inorganic glasses, membranes, or any of the aforementioned supportmaterials (e.g., as a layer or coating).

As used herein, the terms “label” and “tag” refer to substances that mayconfer a detectable signal, and include, but are not limited to, enzymessuch as alkaline phosphatase, glucose-6-phosphate dehydrogenase, andhorseradish peroxidase, ribozyme, a substrate for a replicase such as QBreplicase, promoters, dyes, fluorescers, such as fluorescein,isothiocynate, rhodamine compounds, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine, chemiluminescerssuch as isoluminol, sensitizers, coenzymes, enzyme substrates,radiolabels, particles such as latex or carbon particles, liposomes,cells, etc., which may be further labeled with a dye, catalyst or otherdetectable group.

As used herein, the term “receptor” and “receptor protein” are usedherein to indicate a biologically active proteinaceous molecule thatspecifically binds to (or with) other molecules such as Bcl-2.

As used herein, the term “ligand” refers to a molecule that contains astructural portion that is bound by specific interaction with aparticular receptor protein.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions (fragments) of immunoglobulinmolecules, i.e., molecules that contain an antibody combining site orparatope. The term is inclusive of monoclonal antibodies and polyclonalantibodies.

As used here, the terms “monoclonal antibody” or “monoclonal antibodycomposition” refer to an antibody molecule that contains only onespecies of antibody combining site capable of immunoreacting with aparticular antigen. A monoclonal antibody composition thus typicallydisplays a single binding affinity for any antigen with which itimmunoreacts. A monoclonal antibody composition is typically composed ofantibodies produced by clones of a single cell called a hybridoma thatsecretes (produces) only one type of antibody molecule. The hybridomacell is formed by fusing an antibody-producing cell and a myeloma orother self-perpetuating cell line. Such antibodies were first describedby Kohler and Milstein, Nature, 1975, 256:495-497, the disclosure ofwhich is herein incorporated by reference. An exemplary hybridomatechnology is described by Niman et al., Proc. Natl. Acad. Sci. U.S.A.,1983, 80:4949-4953. Other methods of producing monoclonal antibodies, ahybridoma cell, or a hybridoma cell culture are also well known. Seee.g., Antibodies: A Laboratory Manual, Harlow et al., Cold Spring HarborLaboratory, 1988; or the method of isolating monoclonal antibodies froman immunological repertoise as described by Sasatry, et al., Proc. Natl.Acad. Sci. USA, 1989, 86:5728-5732; and Huse et al., Science, 1981,246:1275-1281. The references cited are hereby incorporated herein byreference.

As used herein, a semi-permeable membrane refers to a bio-compatiblematerial which is impermeable to liquids and capable of allowing thetransfer of gases through it. Such gases include, but are not limitedto, oxygen, water vapor, and carbon dioxide. Semi-permeable membranesare an example of a material that can be used to form a least a portionof an enclosure defining a flow chamber cavity. The semi-permeablemembrane may be capable of excluding microbial contamination (e.g., thepore size is characteristically small enough to exclude the passage ofmicrobes that can contaminate the analyte, such as cells). In aparticular aspect, a semi-permeable membrane can have an opticaltransparency and clarity sufficient for permitting observation of ananalyte, such as cells, for color, growth, size, morphology, imaging,and other purposes well known in the art.

As used herein, the term “bind” refers to any physical attachment orclose association, which may be permanent or temporary. The binding canresult from hydrogen bonding, hydrophobic forces, van der Waals forces,covalent, or ionic bonding, for example.

As used herein, the term “particle” includes insoluble materials of anyconfiguration, including, but not limited to, spherical, thread-like,brush-like, and irregular shapes. Particles can be porous with regularor random channels inside. Particles can be magnetic. Examples ofparticles include, but are not limited to, silica, cellulose, Sepharosebeads, polystyrene (solid, porous, derivatized) beads, controlled-poreglass, gel beads, magnetic beads, sols, biological cells, subcellularparticles, microorganisms (protozoans, bacteria, yeast, viruses, andother infectious agents), micelles, liposomes, cyclodextrins, and otherinsoluble materials.

A “coding sequence” or “coding region” is a polynucleotide sequence thatis transcribed into mRNA and/or translated into a polypeptide. Forexample, a coding sequence may encode a polypeptide of interest. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′-terminus and a translation stop codon at the3′-terminus. A coding sequence can include, but is not limited to, mRNA,cDNA, and recombinant polynucleotide sequences.

As used herein, the term “polypeptide” refers to any polymer comprisingany number of two or more amino acids, and is used interchangeablyherein with the terms “protein”, “gene product”, and “peptide”.

As used herein, the term “nucleoside” refers to a molecule having apurine or pyrimidine base covalently linked to a ribose or deoxyribosesugar. Exemplary nucleosides include adenosine, guanosine, cytidine,uridine and thymidine.

The term “nucleotide” refers to a nucleoside having one or morephosphate groups joined in ester linkages to the sugar moiety. Exemplarynucleotides include nucleoside monophosphates, diphosphates andtriphosphates.

The terms “polynucleotide”, “nucleic acid molecule”, and “nucleotidemolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms. Polynucleotides can encode a polypeptide such as Bcl-2polypeptide (whether expressed or non-expressed), or may be shortinterfering RNA (siRNA), antisense nucleic acids (antisenseoligonucleotides), aptamers, ribozymes (catalytic RNA), ortriplex-forming oligonucleotides (i.e., antigene), for example.

As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acidmolecule” refers generally to a polymer of ribonucleotides. The term“DNA” or “DNA molecule” or deoxyribonucleic acid molecule” refersgenerally to a polymer of deoxyribonucleotides. DNA and RNA moleculescan be synthesized naturally (e.g., by DNA replication or transcriptionof DNA, respectively). RNA molecules can be post-transcriptionallymodified. DNA and RNA molecules can also be chemically synthesized. DNAand RNA molecules can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). Based on the nature of the invention, however, theterm “RNA” or “RNA molecule” or “ribonucleic acid molecule” can alsorefer to a polymer comprising primarily (i.e., greater than 80% or,preferably greater than 90%) ribonucleotides but optionally including atleast one non-ribonucleotide molecule, for example, at least onedeoxyribonucleotide and/or at least one nucleotide analog.

As used herein, the term “nucleotide analog” or “nucleic acid analog”,also referred to herein as an altered nucleotide/nucleic acid ormodified nucleotide/nucleic acid refers to a non-standard nucleotide,including non-naturally occurring ribonucleotides ordeoxyribonucleotides. Preferred nucleotide analogs are modified at anyposition so as to alter certain chemical properties of the nucleotideyet retain the ability of the nucleotide analog to perform its intendedfunction. For example, locked nucleic acids (LNA) are a class ofnucleotide analogs possessing very high affinity and excellentspecificity toward complementary DNA and RNA. LNA oligonucleotides havebeen applied as antisense molecules both in vitro and in vivo (Jepsen J.S. et al., Oligonucleotides, 2004, 14(2):130-146).

As used herein, the term “RNA analog” refers to a polynucleotide (e.g.,a chemically synthesized polynucleotide) having at least one altered ormodified nucleotide as compared to a corresponding unaltered orunmodified RNA but retaining the same or similar nature or function asthe corresponding unaltered or unmodified RNA. As discussed above, theoligonucleotides may be linked with linkages which result in a lowerrate of hydrolysis of the RNA analog as compared to an RNA molecule withphosphodiester linkages. Exemplary RNA analogues include sugar- and/orbackbone-modified ribonucleotides and/or deoxyribonucleotides. Suchalterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA).

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state.

As used in this specification, the singular forms “a”, “an”, and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “an antibody” includes more than onesuch antibody. A reference to “a molecule” includes more than one suchmolecule, and so forth.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology that arewithin the skill of the art. Such techniques are explained fully in theliterature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to MolecularCloning (1984); the series, Methods In Enzymology (S. Colowick and N.Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hameset al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H.Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification Principles and Practice (2nded., Springer-Verlag); and PCR: A Practical Approach (McPherson et al.Eds. (1991) IRL Press)), each of which are incorporated herein byreference in their entirety.

Following are examples that illustrate materials, methods, andprocedures for practicing the invention. The examples are illustrativeand should not be construed as limiting.

Materials and Methods

Patient cohort. With prior institutional approval, urine and bloodsamples were collected from normal healthy control volunteers (N=21),women with benign gynecologic disorders (N=35) and patients with ovariancancer (N=34) at the H. Lee Moffitt Cancer Center. All except 8specimens were collected prior to initial surgical debulking, while thelatter 8 specimens presented with recurrent disease at the time ofenrollment in the study. Paraffin blocks were identified, wherepossible, and the slides reviewed to confirm the histologic diagnosisaccording to FIGO scores. The medical records of these women were alsoreviewed and information regarding patient age, tumor type, stage,grade, size and surgical treatment abstracted where available.

Sample preparation. With patient informed consent, urine and plasmasamples were collected from patients, anonymized and coded to protectpatient identity, and released from the H. Lee Moffitt Cancer Center forthis research protocol. All samples were kept in ice. Urine samples weretreated with a standard protease inhibitor cocktail (80 μg/ml 4-(2aminoethyl)-benzene sulfonyl fluoride, 200 μg/ml EDTA, 0.2 μg/mlleupeptin, 0.2 μg/ml pepstatin, Sigma Scientific, St. Louis, Mich.) andcentrifuged at 3000×g. Urinary supemates and plasma samples were thenaliquoted and stored at −20° C.

Enzyme-linked immunosorbant assay. To measure Bcl-2 levels in patients'urine, samples were assayed using the quantitative sandwichenzyme-linked immunosorbant assay (ELISA; R&D Systems, Minneapolis,Minn.) according to the manufacturer's instructions. To measure CA125levels in subjects' plasma, samples were assayed by ELISA (Bio-Quant,San Diego, Calif.) according to the manufacturer's instructions. Theenzymatic reactions were detected at 450 nm using a Dynex MRX platereader (Dynex Technologies, Chantilly, Va.) and Bcl-2 results expressedas the mean absorbance of triplicate samples±S.E while CA125 resultswere expressed as the mean of duplicate samples.

Statistical analysis. Samples for Bcl-2 ELISA were run in triplicate andthe data subjected to the Kruskal-Wallis test for normal distribution.Data were then analyzed by the Mann-Whitney U-test to determinestatistical significance between samples from normal controls, patientswith benign disease and ovarian cancer patients. Likewise,discrimination analyses using the SAS system were employed to determineappropriate membership in each group (normal vs. benign vs. cancer).

EXAMPLE 1 Urinary Bcl-2 Levels are Elevated in Ovarian Cancer Patients

Urine and blood were collected from 90 individuals with samplescollected from normal controls (N=21), women with benign disease (N=35)and women with ovarian cancer (N=34). The latter category consisted ofwomen diagnosed with endometriod (N=1), mucinous (N=7) as well as serousovarian cancer (N=24) and primary peritoneal cancer, which is oftenrelated to ovarian cancer, (N=2). The samples collected from women withbenign gynecologic disease consisted of women with benign cysticteratomas (N=2), simple cysts (N=10), leiomyomas (N=8), polycysticovarian disease (N=1), ovarian adenofibromas (N=4), mucinouscystadenomas (N=2) and serous cystadenomas (N=8). Though this cohortcomprises a small pilot study, it is representative of a typicalclinical practice with regards to histology, grade and stagedistribution.

To determine the potential suitability of urinary Bcl-2 levels as a newmolecular marker for ovarian cancer, urine samples from the normalcontrols, women with benign gynecologic disease and patients withovarian cancer specimens were screened by ELISA analyses (FIG. 1). Theamount of urinary Bcl-2 was generally negligible (average 0.21 ng/ml) innormal control samples. In contrast, urinary Bcl-2 associated withovarian and primary peritoneal cancer, was generally >10× (3.4 ng/ml)that found in normal control samples (FIG. 1A). No normal urine samplecontained Bcl-2>1.8 ng/ml, while only 2 of the cancer samples exhibitedBcl-2 less than 1.8 ng/ml (1.12 ng/ml and 1.78 ng/ml).

Since serous carcinoma represents the majority of epithelial ovariancancers, urinary Bcl-2 levels in patients with serous adenocarcinomawere examined by disease grade (FIG. 1A) and stage (FIG. 1B). Thoughthere was a tendency for elevated Bcl-2 levels with increasing tumorgrade and stage, the difference in Bcl-2 levels between tumor grade andstage was not statistically significant. Likewise, serum creatinine wasmeasured at time of urine collection and indicated that urinary Bcl-2levels were not related to renal dysfunction (data not shown). Of note,a single patient (#77) demonstrated extremely elevated urinary Bcl-2levels (>9 ng/ml) in the absence of other notable clinical symptoms.

Table 2 summarizes the results presented in FIGS. 1 and 2 for averageBcl-2 levels in urine specimens. Numbers in parentheses indicate thenumber of samples in each respective group. Additionally, the data aregrouped to show average Bcl-2 levels (ng/ml) between normal individualsand ovarian cancer histological subtypes, tumor grade and tumor stage.The data show that while the average level of Bcl-2 in the urine ofhealthy volunteers is 0.204 ng/ml, that from all cancer patients isgenerally 10× greater (3.12 ng/ml). In addition, urinary Bcl-2 levelsappear strongly related to tumor stage and moderately related to tumorgrade among serous ovarian cancers (the most frequently occurring typeof ovarian cancer).

TABLE 2 Urinary Bcl-2 levels in Normal and Ovarian Cancer Sample Bcl-2(ng/ml) Normal (21) 0.204 Endometriod (1) 3.168 Mucinous (4) 2.35Peritoneal (2) 1.78 Serous (29) Grade 1 (7) 2.76 Grade 2 (10) 3.98 Grade3 (12) 3.94 Stage 1 (3) 1.92 Stage 2 (4) 3.23 Stage 3 (14) 4.07 Stage 5(8) 4.04

EXAMPLE 2 Urinary Bcl-2 in Patients with Benign Gynecological Disease isnot Elevated

ELISA measurement of urinary Bcl-2 from 35 women with benign gynecologicdisease (urine collected just prior to patient's treatment) indicatedBcl-2 levels averaging 0.02 ng/ml with no samples>1.8 ng/ml Bcl-2, asshown in FIG. 8A. These benign diseases included benign teratomas,simple cists, leiomyomas, polystronic ovary, fibromas, and adenomas.These values were similar to normal controls, but significantly lessthan ovarian cancer samples suggesting that elevated urinary Bcl-2levels greater than 1.8 ng/ml was associated with ovarian cancer.

The Kruskal-Wallis test was used to test the normal distribution of thedata. Since the ‘normal’ group failed to meet normal distribution,likely due to small sampling number, the differences between groups wereanalyzed by the Mann-Whitney U-test. The results indicated nosignificant difference between normal and benign (p<0.5), but p<0.001between normal and cancer or benign and cancer groups. A summary ofurinary Bcl-2 level for this study group is presented in FIG. 8B.Likewise discrimination analyses using the SAS system revealed that theprobability of appropriate membership in normal/benign or cancer groupwas >90%.

EXAMPLE 3 Urinary Bcl-2 does not Correlate with Patient Age or TumorSize

Comparison of clinical parameters suggested that urinary Bcl-2 levelsdid not relate with patient age (see FIG. 5). Though the age range andaverage age of normal controls (29-81 yr, average 48.5±S.D. 12.7 yr) andwomen with benign gynecologic disease (28-84 yr, average 55.9±S.D. 13.9yr) was somewhat lower that that of women with ovarian cancer (26-92 yr,average 62.2±S.D. 13.8 yr), the differences were not statisticallysignificant in this study. Similarly, urinary Bcl-2 levels did notcorrelate with tumor size measured at debulking surgery (FIG. 6),ranging from microscopic to >10 cm and may reflect biologic variationbetween individuals or variation of tumor composition.

EXAMPLE 4 Urinary Bcl-2 detects ovarian cancer more accurately thanCA125 in blood

To address whether elevated urinary Bcl-2 is a better diagnosticindicator for ovarian cancer than cancer antigen 125 (CA125), urinaryBcl-2 was compared with CA125 levels in 12 normal controls and 23patients with ovarian cancer (FIGS. 4A and 4B). Of the patientsexamined, elevated urinary Bcl-2 associated with ovarian cancerdetection was almost 100%. Elevated urinary Bcl-2 (>1.8 ng/ml)identified 17/17 patients with serous adenocarcinoma, 4/4 patients withmucinous ovarian cancer and ½ patients with primary peritoneal cancer asovarian cancer positive (FIG. 4A). None of the normal controls hadurinary Bcl-2 levels>1.8 ng/ml and were, then, correctly classified ascancer-negative. In contrast, blood levels of CA125>35 U/ml, the currentstandard for ovarian cancer detection, identified 13/17 or 76% ofpatients with serous adenocarcinoma (FIG. 4B). Likewise, CA125 analysesidentified ¾ or 75% of patients with mucinous ovarian cancer, thoughCA125 levels in these patients ranged between 41-43 U/ml, and ½ or 50%of patients with primary peritoneal cancer as cancer positive. ElevatedCA125 levels also incorrectly identified 2/12 or 16% of healthyindividuals as cancer-positive suggesting that elevated urinary Bcl-2appears to detect ovarian cancer more accurately than CA125.

EXAMPLE 5 Urinary Bcl-2 Decreases After Debulking Surgery

To further test the accuracy for high levels of urinary Bcl-2 to detectovarian cancer, levels of urinary Bcl-2 were compared in 7 ovariancancer patients immediately prior to (FIGS. 7A and 7B, black bars) andwithin 2 weeks following initial debulking surgery for removal of allvisible tumor (white bars). For those patients where urine samples werecollected before and after initial surgery, Bcl-2 levels decreased up to100% following surgical removal of tumor suggesting that presence oftumor correlates well with elevated urinary Bcl-2 in ovarian cancerpatients.

Currently, preclinical studies focus on the development of agents toinhibit Bcl-2, including antisense oligonucleotides and small molecularinhibitors of Bcl-2. Though such studies target Bcl-2 for therapeuticintervention, the present data indicate that quantification of urinaryBcl-2 by ELISA-based assays may provide a novel, safe, sensitive,specific and economical method for the detection of ovarian cancer thatwould benefit all women not only in the US, but worldwide includingmedically underserved geographical areas and especially women at highrisk for developing ovarian cancer. Further, given that approximately25,000 women are diagnosed with ovarian cancer annually in the US,urinary Bcl-2 detection of ovarian cancer in both early and late stagesof disease would not only confirm the diagnosis of ovarian cancer, butcould also potentially detect thousands of previously undiagnosedovarian cancers. This is especially important for detection of ovariancancer in early stages that account for less than 10% of diagnosedovarian cancers, but where surgical debulking of the diseased ovaryincreases patient survival to over 90% and would be expected to reducelife long medical costs. Lastly, in addition to serving a noveldiagnostic function, urinary levels of Bcl-2 can be used to monitor thepresence of ovarian cancer throughout the course of disease which mayimpact therapeutic and prognostic outcome. Clearly, larger populationstudies are warranted to verify the potential for urinary levels ofBcl-2 to serve as a biomarker for ovarian cancer as well asinvestigations into the molecular mechanism(s) responsible for elevatedurinary Bcl-2 in ovarian cancer. However, since there are no reportsthat employ either urinary detection or Bcl-2 as a biomarker for ovariancancer, this pilot study suggests that measurement of urinary Bcl-2 byELISA may provide an innovative, simple method to detect all ovariancancers and, possibly, reduce the mortality of an insidious disease thatkills thousands of women annually.

EXAMPLE 6 Urine Storage Conditions for Bcl-2 Testing

Studies examining the storage stability of urinary bcl-2 indicate thatwhen samples are prepared with the addition of a cocktail of proteaseinhibitors these urine samples may be stored for over 1 year at −20° C.without loss of bcl-2 detection (see ‘Control’ & ‘−20° C.’ in FIG. 11).This would be beneficial for individuals where it might be desirable tore-test previous samples with current ones. Alternately, these samplescan also be stored at 4° C. for up to 4 days without adversely affectingdetection of urinary Bcl-2. These are important results as they indicatethat the time possibly required to transport patient urine samples (frompotentially distant geographical areas) to a laboratory for Bcl-2testing would not adversely affect the outcome of urinary bcl-2detection if protease inhibitors are added to the urine samples and theurine samples are kept cold. However, reduced Bcl-2 was measured insamples stored at room temperature for 4 days and Bcl-2 could not bedetected in urine samples stored at −80° C.; therefore, it appearsprohibitive to store urinary samples for Bcl-2 detection at either roomtemperature or at −80° C.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method of accurately screening for ovarian cancer in a subject,comprising detecting the presence of Bcl-2 protein in a biologicalsample of urine from the subject using ELISA-based immunoenzymaticdetection; and determining whether the level of Bcl-2 protein in thesample exceeds a predetermined threshold representing the level ofurinary Bcl-2 protein in a non-cancerous individual, wherein a level ofBcl-2 protein above the pre-determined threshold level of urinary Bcl-2protein of a non-cancerous individual is accurate to distinguish asubject having ovarian cancer from a non-cancerous individual.
 2. Themethod of claim 1, wherein said detecting comprises: (a) contacting thebiological sample with a binding agent that binds Bcl-2 protein to forma complex; (b) detecting the complex; and (c) correlating the detectedcomplex to the amount of Bcl-2 protein in the sample.
 3. The method ofclaim 2, wherein the binding agent is immobilized on a support.
 4. Themethod. of claim 2, wherein the binding agent is a monoclonal orpolyclonal antibody.
 5. The method of claim 2, wherein said detecting of(b) further comprises linking or incorporating a label onto the bindingagent.
 6. The method of claim 1, further comprising detecting abiomarker of cancer other than Bcl-2 in the same biological sample or adifferent biological sample obtained from the subject, before, during,or after said detecting of Bcl-2.
 7. The method of claim 6, wherein thebiomarker of cancer is a biomarker of gynecological cancer.
 8. Themethod of claim 6, wherein the biomarker is CA125, LPA, or OVXI.
 9. Themethod of claim 1, wherein said subject is suffering from cancer, andwherein said detecting is performed at several time points at intervals,as part of a monitoring of the subject before, during, or after thetreatment of the cancer.
 10. The method of claim 1, further comprisingcomparing the level of Bcl-2 protein in the subject's biological samplewith the level of Bcl-2 protein present in a normal control sample ofurine from a non-cancerous individual, wherein a higher level of Bcl-2protein in the subject's biological sample as compared to the level inthe normal control sample is indicative of cancer.
 11. The method ofclaim 1, wherein the subject is exhibiting no symptoms of cancer at thetime said detecting is carried out.
 12. The method of claim 1, whereinthe subject is exhibiting one or more symptoms of cancer at the timesaid detecting is carried out.
 13. The method of claim 1, whereinsubject is exhibiting one or more of the symptoms selected from thegroup consisting of pelvic pain, abnormal vaginal bleeding, abdominalswelling or bloating, persistent back pain, persistent stomach upset,change in bowel or bladder pattern, pain during intercourse,unintentional weight loss of ten or more pounds, vulva or vaginalabnormality, change in the breast, and fatigue.
 14. The method of claim1, wherein the subject has an elevated CA125 level in the urine at thetime of said detecting.
 15. The method of claim 1, wherein the subjectdoes not have an elevated CA125 level in the urine at the time of saiddetecting.
 16. The method of claim 1, wherein said predeterminedthreshold is between 0.0 ng/ml and 2.0 ng/ml.
 17. The method of claim 1,wherein said predetermined threshold is 1.8 ng/ml.